Download Transcription factor AP-2 is tissue-specific in

283
Development 113, 283-293 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Transcription factor AP-2 is tissue-specific in Xenopus and is closely
related or identical to Keratin Transcription Factor 1 (KTF-1)
ALISON M. SNAPE, ROBERT S. WINNING and THOMAS D. SARGENT*
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland 20892, USA
* Author for correspondence
Summary
This paper identifies a new, developmental role for
transcription factor AP-2 in the activation of amphibian
embryonic epidermal keratin gene expression. Keratin
transcription factor KTF-1 is shown by several criteria
to be identical or closely related to AP-2. KTF-l/AP-2 is
shown to be tissue-specific from its first transcription in
Xenopus embryos, and restricted to a small number of
adult tissues, including skin. Epidermis-specific keratin
transcription closely follows specification of the embryonic ectoderm in Xenopus, and is subject to regulation by
growth factors and embryonic induction. We further
show that in mouse basal keratinocytes, a KTF-l/AP-2like factor is present and binds to a DNA sequence
previously shown to be important in the regulation of the
keratin K14 gene, which is actively expressed in these
cells. Thus, the study of AP-2 and its role in the
regulation of keratin gene transcription should enhance
our understanding of both amphibian embryonic development and mammalian skin differentiation.
Introduction
underlying dorsal mesoderm (Symes et al. 1988; Dawid
et al. 1988; Jamrich et al. 1987). A knowledge of the
factors controlling tissue-specific expression and repression of XK81A1 could, thus, lead to the identification of autonomous ectodermal determinants and a
greater understanding of the molecular mechanisms of
embryonic induction.
In earlier studies, we showed that 487 base pairs (bp)
of 5' flanking sequence were sufficient to drive
epidermis-specific transcription from the XK81A1 gene
injected into Xenopus embryos (Jonas et al. 1989).
Subsequently, we identified a protein-binding positiveregulatory sequence within this promoter region,
mutation of which reduced epidermal expression of the
injected gene by up to 90% (Snape et al. 1990). (We
recently learned that in these expression studies we
erroneously used as a control DNA a construction with
275 rather than 487 bp of 5' flanking DNA. We have
repeated these expression studies using the correct
control, with essentially identical results.) Nuclear
extracts prepared from Xenopus embryos contained a
protein, designated KTF-1 (for Keratin Transcription
Factor-1), which bound specifically to this positiveregulatory sequence. We were unable to purify KTF-1
protein directly from embryos in quantities sufficient
for protein sequence, determination. However, as
described below, we were able to exploit similarity
Xenopus embryonic epidermis is an ectodermal tissue
that derives from the animal cap of the pre-gastrula
embryo. Like other epithelia, the epidermis expresses
an array of keratins which constitute a major component of the cytoskeleton. The keratin family of
intermediate filament proteins contains at least twenty
polypeptides, which are differentially expressed in
patterns typical of the developmental origin and stage
of the tissue. Thus, the study of keratin expression can
yield insight into the mechanisms of epithelial differentiation. In Xenopus, the gene for the embryonic keratin
XK81A1 is especially interesting, because it is transcribed immediately following the midblastula transition (MBT), specifically in the animal cap (Jamrich et
al. 1987). Furthermore, embryonic blastomeres will
autonomously turn on XK81A1 at the appropriate time
when dispersed in culture (Sargent et al. 1986; Symes et
al. 1988), indicating that the determinants necessary to
specify ectoderm are already localized in the animal cap
at very early stages. Initially, all the cells of the
ectoderm express XK81A1, but transcription can be
repressed in response to a number of inductive stimuli,
including the conversion of ectoderm to mesoderm by
growth factors such as activin A (XTC-MIF), and, after
gastrulation, the induction of the neural plate by
Key words: Xenopus, embryo, keratin, transcription factor,
AP-2.
284
A. M. Snape, R. S. Winning and T. D. Sargent
between the KTF-1 binding sequence, and that for the
human transcription factor AP-2, to take an alternative
approach to the identification of KTF-1.
AP-2 is an enhancer-binding protein which has been
purified and cloned from HeLa cells, and specifically
interacts with the consensus sequence GCC( / C )G( C /
G )GGC (Mitchell et al. 1987,1991; Williams etal. 1988).
The AP-2 consensus binding sequence matches the
central core of the KTF-1 binding site (CCCTGAGG)
at 7 out of 9 positions. AP-2 sites are found in the
promoters of a number of viral and cellular genes and,
in many cases, these sites are important in regulating
transcription. There is evidence supporting a role for
AP-2 in early developmental decisions; several genes
that are activated in response to retinoic acid-induced
differentiation of mammalian teratocarcinoma cells
contain AP-2 sites (summarized in Liischer et al. 1989).
Recently, Mitchell et al. (1991) have shown tissuespecific distribution of AP-2 RNA in the mouse
embryo, including expression in 'pre-epidermis'. As
part of a project to identify transcription factors that
might play a role in early amphibian development, the
Xenopus cDNA homologous to human AP-2 has
recently been cloned (Winning et al. 1991). We now
show that the protein encoded by the Xenopus-AP-2
(XAP-2) cDNA shares many properties with KTF-1,
and conclude that XAP-2 is, in fact, identical or closely
related to KTF-1. We show that KTF-l/XAP-2 RNA
and DNA-binding activity are localized in the epidermis
of the Xenopus embryo, consistent with a role for this
transcription factor in the regulation of keratin expression. We also present evidence supporting the
suggestion, made by Leask et al. (1990), that a
transcriptional activator of the mammalian basal
epidermal cell-specific keratin, K14, is AP-2 or an AP2-like protein, suggesting evolutionary conservation of
the regulation of mammalian K14 and the closely
related amphibian keratin gene, XK81A1. Transcriptional activation via mammalian AP-2 can be induced
by retinoic acid (Liischer et al. 1989), cAMP and
phorbol esters (Imagawa et al. 1987), or repressed by
SV40 large T antigen (Mitchell et al. 1987). Thus,
several developmental^ important agents may act via
KTF-l/XAP-2 in the control of embryonic epidermal
gene expression.
Besides showing tissue-specific embryonic expression, KTF-l/XAP-2 RNA and protein were found
to be expressed specifically in skin, kidney and brain of
the adult frog. By analogy with the human K14 gene,
KTF-l/XAP-2 may be involved in transcriptional
regulation of certain adult keratin genes in Xenopus
skin, but, like mammalian AP-2, it probably also
regulates non-keratin genes in Xenopus embryos and
adults
Materials and methods
Materials
Xenopus laevis embryos were obtained by artificial fertilization, as in Jonas et al. (1989) and maintained in dechlorinated
tap water until they reached the desired stage. Metamorphosing tadpoles and adult frogs were purchased directly from
Nasco.
Whole cell extracts
These were prepared by the method of Kumar and Chambon
(1988) in buffer containing 0.6 M KC1, with the modification
that protease inhibitors were added to the buffer as follows:
PMSF (Sigma) at lmM, aprotinin at 28/igml"1 and leupeptin
at 14/igml"1 (both United States Biochemical). Xenopus
embryos were homogenized on ice in 300-500/xl buffer per
100 embryos in a Dounce glass homogenizer (B pestle).
Metamorphosing tadpoles and adult tissues were blended for
30 s, in approximately 3 ml buffer per tadpole or per gram of
tissue, using a Tekmar Tissumizer, and filtered through
cheesecloth before homogenizing. To allow more extract to
be added to binding reactions without raising the salt
concentration above 200 HIM (the upper limit for maximal
KTF-1 binding), the extract from metamorphosing tadpoles
was dialysed against buffer containing 0.2 M KC1. Extracts
were prepared from cultured mouse basal epidermal cells (the
gift of S.Yuspa; Yuspa et al. 1989) and WI-38 cells (the gift of
T. Howard) by the freezing method of Kumar and Chambon
(1988), using 1 ml buffer per 150 cm2flaskof cells. The protein
concentration of extracts was determined using a Pierce BCA
protein assay.
In vitro translated XAP-2
This was prepared as in Winning et al. (1991), except that the
protein was not radioactively labeled.
DNA-mobility shift assays
These were carried out as in Snape et al. (1990) with the
following modifications. (1) The binding buffer was prepared
without NaCl, since salt was supplied by the extract buffer. (2)
The reaction volume in some experiments was increased to
40^1, to allow for the addition of a greater volume of extract.
Routinely, 15-20^1 metamorphosing tadpole extract, 4^1
tissue culture cell extract and 2\A in vitro translated XAP-2
were used per binding reaction. Where necessary, extract
buffer was added to the reaction to equalize salt concentrations within an experiment. (3) Poly (dI-dC)(dI-dC) nonspecific competitor (Pharmacia) was increased to 7.5 or 10,ug
per reaction.
Double-stranded oligonucleotides for probes and competitors were synthesized according to the sequences given in
Fig. 4, with 4 bases (TCGA) added to the 5' end of each one,
to create overhanging ends when the complementary strands
were annealed. To make labeled probe, the ends werefilledin
using the Klenow fragment of DNA polymerase I (BRL) and
[32P]TTP (Amersham) as in Ausubel etal. (1987). Unincorporated nucleotides were removed by isolating the probe
from a 10% acrylamide gel (Ausubel et al. 1987). For the
DNA-sequence binding-specificity analysis, the intensity of
shifted bands was measured on a Macbeth X-ray film
densitometer, using films exposed within the linear response
range.
Proteolytic clipping bandshift assays
These were based on the method of Schreiber et al. (1988).
After incubation of binding reactions as above, 1 fi\ diluted
protease (Boehringer Mannheim) was added to each reaction
and allowed to react for lOmin at room temperature, before
electrophoresis as for the standard DNA-mobility shift assay.
Antibody binding reactions
These were carried out as follows: standard DNA-binding
AP-2 and KTF-1 are related or identical
reactions were set up as above, without probe DNA or
competitor dl-dC, and 2.5 ^il (antiserum to DNA-binding
region, and anti-Ha-ras) or 1^1 (antiserum to carboxyl
terminal region) was added and allowed to react for 1 h on ice.
Probe and dl-dC were then added, and after further
incubation for 15min at room temperature, electrophoresis
was carried out as usual. Control affinity-purified rabbit antihuman Ha-ras was purchased from Oncor (Gaithersburg,
MD).
tadpole
extract
KTF-1
Competitor:
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285
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XAP-2
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Tissue specificity analysis of Xenopus embryos
Dissections were carried out in 100 min NaCl, 2 mM KC1, 1 HIM
MgSO4, 2mM CaCl2, 5mM Hepes (pH7.0), 0.1 mM EDTA.
Blastulae were divided into animal (including all the
pigmented cells) and vegetal hemispheres, using watchmakers
forceps. Epidermis was removed from tailbud stages by
drawing the embryo into and expelling it from a drawn-out
Pasteur pipette. The epidermis was then cleaned in dissociating 67 mM sodium phosphate, pH7.6. All the cells of each
embryo were collected, as far as possible. For each stage, 110
embryos were dissected. Nucleic acid was isolated from 10
dissected and 10 whole embryos by the method of Sargent et
al. (1986), except that LiCl precipitation was omitted, so that
both RNA and DNA were retained. Whole cell extracts were
prepared from 100 dissected and 20 whole embryos. To
estimate the number of cells in each fraction, DNA dot blots
(Jonas et al. 1989) were hybridized with a nick-translated
probe specific for 1723, a highly repeated, interspersed
element in the Xenopus genome (Kay and Dawid, 1983) and
counted on an Ambis gel scanner. The volume of RNA or
extract was adjusted according to this estimation, so that
material from the same number of cells was used in each lane
or DNA-binding reaction.
RNA gel blots
RNA from adult tissues, isolated as in Sargent et al. (1986),
was the gift of V. Agarwal. RNA gels were run, blotted and
hybridized with nick translated probes as in Sargent et al.
(1986). Integrity of the RNA was confirmed by ethidium
bromide staining of the gel before blotting. The probes used
were a 1000 bp EcoRI fragment from a XAP-2 cDNA clone,
XAP2-6a (Winning et al. 1991), or an EcoRI-Ncol fragment
from a human AP-2 cDNA clone, AP-2Rl/Nco, the gift of
T.Williams. Probes were labeled using a nick-translation kit
from BRL, and [32P]dCTP from Amersham.
Fig. 1. DNA-mobility shift assay comparing binding of in
vitro translated XAP-2 protein and tadpole extract to an
oligonucleotide probe containing the KTF-1 binding site
from the XK81A1 keratin gene. For probe and unlabeled
competitor oligonucleotide sequences see Fig. 4.
32
P-labeled probe is X-157. Unlabeled competitors were
added to 1000-fold the concentration of the labeled probe.
Tadpole extract KTF-1 is whole cell extract prepared from
metamorphosing Xenopus tadpoles (see Materials and
methods). The lowest band in each lane represents
unbound probe.
Results
KTF-1 and AP-2 are identical or closely related
proteins
DNA-mobility shift assay
Previously (Snape et al. 1990), we noted sequence
homology between the XK81A1 KTF-1 site, and the
binding site for the human transcription factor AP-2. As
this laboratory has recently cloned the cDNA corresponding to the Xenopus homologue of human AP-2
(Winning et al. 1991), we decided to investigate the
possible relationship between KTF-1 and XAP-2 by
comparison of the properties of the two proteins.
Winning et al. (1991) have shown that in a DNAmobility shift assay, in vitro translated XAP-2 binds
specifically to an AP-2 site in the promoter of the
human metallothionein gene. Fig. 1 shows that both
XAP-2 and KTF-1 specifically bind to a 23 base pair
(bp) XK81A1 promoter sequence containing the KTF-1
site, but not to a control mutated oligonucleotide. This
shows that XAP-2 can recognise the same binding site
as KTF-1, and may be a related protein. In the
experiment shown in Fig. 1, the mobility of the
DNA-protein complex is slightly different for KTF-1
and XAP-2, suggesting that the proteins may not be
completely identical. However, the difference might
alternatively be due to post-translational modification
of the protein, which does not take place in the in vitro
translation system, to complex formation between
KTF-1 and another protein in the tadpole extract, or to
proteolysis of in vitro translated XAP-2. Furthermore,
the mobility of the complex obtained using embryo
extracts was somewhat variable, and in other experiments (not shown) it appeared more similar to that
obtained with XAP-2.
286
A. M. Snape, R. S. Winning and T. D. Sargent
A. Xenopus tadpole KTF-1
Endoproteinase
ArgC
_
TrypSI
.
"
Endoproteinase
GluC
1
B. in vitro translated XAP-2
Endoproteinase
Trypsjn
Endoproteinase
•to
» I
Fig. 2. Proteolytic clipping bandshift assays comparing KTF-1 from tadpole extracts and in vitro translated XAP-2. The
DNA mobility shift probe is X-157 (see Fig. 4). Proteinases were added to the binding reactions before electrophoresis, in
1
the following concentrations (lanes from left to right): Endoproteinase
Arg
0,
p
g C;
; 0,, 0.09,
, 0.9,
, 9 U^l"
^ ; ; Trypsin;
yp;
, 0.9,
, 3, 9,
1
11
30 ngjid" ; Endoproteinase Glu C; 0, 0.3, 3,
1
. The lowest
band in
each laneasrepresents
probe.
The
mobility of the band shifted due to KTF-1 or XAP-2 binding
is gradually
increased,
the proteinunbound
is digested
by increasing
proteinase concentrations. (A) Whole cell extract from metamorphosing Xenopus tadpoles. Extract from tailbud (stage
19-24) embryos gave the same pattern (not shown). (B) In vitro translated XAP-2.
Proteolytic clipping bandshift assays
Further evidence that KTF-1 and XAP-2 are related
proteins was obtained using the proteolytic clipping
bandshift assay (PCBA) technique (Schreiber et al.
1988), as shown in Fig. 2. DNA-binding reactions
containing the KTF-1 binding site probe, and either in
vitro translated XAP-2 or Xenopus whole cell extracts,
were treated with increasing concentrations of three
proteases, each of which has a different cleavage
specificity. When the reactions were run on a nondenaturing acrylamide gel, under conditions routinely
used in the mobility-shift assay, each protease gave a
characteristic pattern of bands with higher mobility
than the undigested protein-DNA complex, resulting
from partial digestion of the DNA-binding protein.
Only the peptide fragment containing the DNA-binding
domain is detected in this assay. With each protease,
the pattern obtained for XAP-2 (Fig. 2B) was identical
or closely similar to the patterns obtained for Xenopus
extracts from either metamorphosing tadpoles
(Fig. 2A) or tailbud (stage 19-24) embryos (data not
shown).
Antibody-binding reactions
Samples of two rabbit antisera, raised against different
regions of human AP-2, were the gift of T. Williams
(laboratory of R. Tjian). Evidence that these antibodies
cross-react with KTF-1 was obtained by incubating the
antisera with extract from metamorphosing Xenopus
tadpoles and then adding the KTF-1 binding site probe.
When the reactions were run on a mobility-shift assay
gel (Fig. 3), there was a small, but reproducible,
reduction in the intensity of the shifted band in the
reaction with antiserum to the DNA-binding region,
suggesting that binding of the antibody to KTF-1
prevented access of the DNA probe to its binding site.
In the reaction with antiserum to the carboxyl-terminal
region, the migration of the DNA-protein complex was
obviously, and reproducibly, retarded, suggesting that
this antibody also cross-reacts with KTF-1 and alters the
mobility of the DNA-protein complex, but does not
prevent KTF-1 from binding DNA. A control, affinitypurified antiserum, anti-human Ha-ras, neither reduced
the intensity nor altered the mobility of the KTF-1
shifted band (Fig. 3). Thus, Xenopus KTF-1 shares
epitopes with human AP-2.
DNA-binding specificities of KTF-1 and XA P-2
A comparison of the DNA-binding specificities of in
vitro translated XAP-2 and KTF-1 from tadpole
extracts was carried out. Besides the KTF-1 site at -157
(oligonucleotide X-157; see Fig. 4), the XK81A1
promoter contains a similar sequence at -254 (oligonucleotide X-254), which is conserved in related or coregulated Xenopus embryonic keratin genes (Fig. 4).
Moreover, the promoter of K14, the human keratin
with the closest known homology to XK81A1, contains
two AP-2 like sites (oligonucleotides K14-229 and
K14-86), one of which, at -229, can bind a nuclear
protein from mouse keratinocytes (see Fig. 9), and act
as a positive control element for K14 transcription in
human keratinocytes (Leask et al. 1990). Several of the
AP-2 and KTF-1 are related or identical
O5
c
•§
z n
* ^
nti •Ha-r
o
Antibody:
(A
(0
nti •Ctei
I
Fig. 3. DNA-mobility shift assay showing that antisera
raised against human AP-2 cross-react with KTF-1. Whole
cell extract from metamorphosing tadpoles was preincubated with antisera before addition of the labeled
X-157 oligonucleotide probe, followed by electrophoresis.
The lowest band represents unbound probe. The band
shifted due to KTF-1 binding shows either reduced
intensity due to binding of antibody to the DNA-binding
domain, or retarded migration due to binding of antibody
to the carboxyl terminus. Control anti-Ha-ras had no effect
on the shifted band.
w
3
'"& C
'2c5 E
(0
(0
3
followed by K14-229, MT-175 (the AP-2 site from the
human metallothionein gene basal element), and
X-254. The K14-86 oligonucleotide competed poorly.
None of the three control mutated oligonucleotides
competed to any appreciable extent for KTF-l/XAP-2
binding (Fig. 5).
1
i
potential KTF-l/AP-2 sites illustrated in Fig. 4 were
used as competitors in the mobility-shift assay. The
affinities of XAP-2 and tadpole KTF-1 for these
competitors were very similar (Table 1 and Fig. 5),
suggesting that the two proteins are structurally closely
related, at least at the DNA-binding domain. For both
factors, the most efficient competitor was X-157,
KTF-1 and XAP-2 are tissue-specific, with similar
expression profiles
This laboratory has already demonstrated that XAP-2
RNA is absent from Xenopus oocytes and cleavagestage embryos, with expression beginning shortly after
the MBT (Winning et al. 1991). If the KTF-1 DNAbinding activity followed a similar time course, this
would be compatible with XAP-2 being KTF-1, and
playing an important role in keratin expression in the
embryonic epidermis. Whole cell extracts were, therefore, prepared from staged embryos, and mobility-shift
assays carried out using the KTF-1 (X-157) probe
(Fig. 6A). A shifted band was observed using extracts
from pre-MBT embryos, but neither unlabeled X-157
nor control mutated X-157M oligonucleotides at 1000fold excess competed for binding, indicating that this
band represents non-specific binding (Fig. 6B). Specific
KTF-1 activity is absent from embryos until after the
MBT; it is first detectable at stage 9, corresponding well
with the beginning of XAP-2 transcription, which takes
place between stages 8 and 9 (Winning et al. 1991). The
specificity of the shifted band observed using extract
from gastrula-stage embryos is shown in Fig. 6B. The
level of KTF-1 activity remains approximately constant
from stage 11 throughout embryogenesis (stages 19, 22,
28 and 35/36 were also assayed, but are not shown),
Table 1. Mobility shift competition data
Extract
Competitor
Mouse basal cell
In vitro XAP-2
Tadpole KTF-1
X-157
K14-229
MT-175
X-254
K14-86
287
5x
50 x
500 x
5x'
50 x
48/69
9/5
23/0
20/0
23/2
90/93
63/62
55/14
25/0
0/0
93/95
91/95
87/74
66/50
61/17
57/50
ND/0
49/62
28/17
ND/32
97/97
NS/67
60/64
32/0
ND/14
500 x
5x
50 x
500 x
99/100
ND/97
96/95
88/78
NS/60
85/65
2/3
45/0
0/7
24/0
99/99
53/23
14/5
14/0
14/0
100/100
98/92
84/60
61/30
58/17
The values correspond to the percent reduction in the mobility shift of a radiolabeled X-157 probe observed when varying amounts of
unlabeled oligonucleotides were added as competitors. Complete competition is denoted by 100, and failure to observe competition is
denoted by 0. ND, not done. None of the control mutated oligonucleotides exhibited significant competition, and these values are not
shown. The results of two independent experiments are separated by the slash mark.
288
A. M. Snape, R. S. Winning and T. D. Sargent
gene
A)
Xenopus keratins
position
XK81A1
XK81A1
XK81B1
XK81B2
XK70A
-157
-254
-248
-240
-269
human keratin
K14
K14
-86
-229
human metallothionein
hMT-ll.
-175
XK81A1
XK81A1
hMT-ll A
-157
-254
-175
sequence
competitor
name
AACAAACACCCTG AGGCTACGTA
TGCTGAAGGAAaCCTGaAGcaaGGAGAGAG
tqCCTGqgGcaq
tgCCTGqqGcaa
caCCTG AaGta
X-157
X-254
TGGCTTTCATCACCCac AGGCTAGCGCCAACT
GGGAAAGTGTAaCCTGcAGGCcCACACCTCCC
GAACTGACcoCCcG C G G C C C G T G T G C A G A G
K14-86
K14-229
MT-175
B)
mutated sequences
AACAAACAtttca aaatTACGTA
X-157 M
TGCTGAAGGAAt at cGat t caaGGAGAGAG
X-254M
GAACTGACctttca aaatcCGTGTGCAGAG MT-175M
Fig. 4. (A) Sequences of putative KTF-l/(X)AP-2 binding sites. In XK81A1 the underlined bases make up an 11 bp
imperfect palindrome, around a central G at -157. The position of the other sites is given as the position of the base
equivalent to that G. Bases in bold type correspond to those making up an AP-2 site, as defined by Mitchell et al. (1991).
Within the binding site, bases in lower case differ from those at equivalent positions in the XK81A1 -157 site. In some
cases, a gap has been left in the sequence, to facilitate alignment of the sites. Where the site was used as a probe or
competitor in DNA-mobility shift assays, additional promoter sequence, outside the binding site, was included in the
oligonucleotide; these sequences are also given. References for these sequences are as follows: XK81A1, Bl and B2,
Miyatani et al. (1986); XK70A, Krasner et al. (1988) and GenBank accession no. M59455; K14, Marchuk et al. (1985);
hMT-IIA, Karin and Richards (1982). (B) Sequences used as mobility shift competitors, corresponding to promoter
sequences with bases altered so as to eliminate KTF-l/(X)AP-2 binding.
followed by an increase at around the feeding tadpole
stage (stage 45). Thus the developmental profiles of
KTF-1 protein and XAP-2 RNA are similar, consistent
with the XAP-2 gene encoding KTF-1.
It was also interesting to find out whether KTF-1
DNA-binding activity and accumulation of XAP-2
RNA corresponded with respect to tissue specificity.
Therefore, whole cell protein extracts and total RNA
were prepared from late-blastula-stage Xenopus embryos dissected into animal (pigmented) and vegetal
(non-pigmented) halves, tailbud-stage Xenopus embryos dissected into epidermal and non-epidermal
fractions, and a selection of adult Xenopus tissues. For
each source, RNA gel blots were probed for XAP-2
RNA, while mobility shift assays for KTF-1 were
carried out using whole cell extracts (Figs 7 and 8).
When assaying KTF-1 DNA-binding activity in
dissected embryos (Fig. 7A), the genomic DNA content of each fraction was compared (see Materials and
methods) and the volume of extract added to the DNAbinding reactions was modified accordingly, so that
each contained extract from an equivalent number of
cells. At the blastula stage there was enrichment of
KTF-1 in the animal half, from which the epidermis,
plus other tissues, derives and, by the tailbud stage,
KTF-1 activity was strikingly localized in the epidermal
A)
Competitors:
X-157
X-157 M
X-254
X-254"
K14-229
K14-86
MT-175
MT-175*
100—.
Tadpole
Extract
KTF-1
5 50 500
550500
5 50 500
550500
5 50 500
5 50 500
S 50 500
5 SO 500
55OSO0
55OSOO
5 SO SOO
550500
550500
550500
B) \
10090-
Transtated
XAP-2 .
Fold Excess Competitor
Jl
550500
S 50 500
Fig. 5. Bar charts illustrating DNA-sequence
binding specificity of KTF-1 from
metamorphosing tadpole extracts (A) and in
vitro translated XAP-2 (B). Unlabeled
oligonucleotides were used in DNA-mobility
shift assays as competitors for binding to the
32
P-labeled X-157 probe, at 5-, 50- and 500times the probe concentration. In each case,
the intensity of the shifted band was
compared to the intensity of the band shifted
when no oligonucleotide competitor was
added. Results are plotted on the vertical axis
as percentage competition, i.e. 100%
competition indicates that there was complete
elimination of the shifted band. Each result is
the mean of two experiments; the data for
experimental competitors are presented in
Table 1.
AP-2 and KTF-1 are related or identical
B.
A.
Stage 1 Stage 10
1 6 8.5 9 10 11 13 17
45
E
A.
Stage 9 Stage 19-24
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Stage:
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Fig. 6. (A) DNA-mobility shift assay showing time course
of KTF-1 DNA-binding activity during Xenopus embryonic
development. The probe is X-157. Whole cell extract was
prepared from 100 embryos of each stage shown
(Nieuwkoop and Faber, 1967), and extract equivalent to
that from 2.5 embryos was used in each reaction. The
lowest band represents unbound probe. The open arrow
indicates probe shifted by a non-specific binding reaction.
The closed arrow indicates probe shifted by specific KTF-1
binding. (B) Competition analysis showing non-specific
DNA-binding activity from stage 1 embryos (fertilized
eggs), not competed efficiently by oligonucleotide probes
(open arrow) versus specific KTF-1 activity (closed arrow)
from stage 10 embryos (gastrulae), competed by 1000-fold
excess of unlabeled probe (X-157) oligonucleotide, but not
by 1000-fold excess of X-157M, the equivalent sequence
with the KTF-1-binding site mutated.
fraction. When the RNA gel blot (Fig. 7B) was loaded
in the equivalent fashion (RNA from an equal number
of cells in each lane), XAP-2 transcripts were localized
in the blastula animal half, and the tailbud epidermis.
Once again, the evidence is consistent with the
hypothesis that XAP-2 is KTF-1.
In adult Xenopus tissues, both KTF-1 activity and
XAP-2 RNA show similar, highly tissue-specific,
expression patterns (Fig. 8). In the KTF-1 assay, the
volume of extract in the DNA-binding reactions was
modified so that equal amounts of total protein were
compared for each tissue. Since many of the extracts
appeared negative for KTF-1, the experiment was then
repeated with the maximum possible volume of extract
in each reaction. Still, KTF-1 activity could not be
detected in ovary, liver or lung. Adult skin and kidney,
however, both contained significant KTF-1 activity.
When total RNA was probed for XAP-2 RNA,
transcripts were seen in skin and kidney, but not in
oocyte, liver or lung RNA. This is further strong
evidence that KTF-1 and XAP-2 are the same factor. In
addition, testis RNA is negative and brain RNA is
weakly positive for XAP-2 transcripts. Protein extracts
were not prepared from these tissues, so there are no
data on their KTF-1 activity. Since adult Xenopus
tissues (except possibly the esophageal lining; Fouquet
et al. 1988) do not express the embryonic keratin genes
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RNA gel blot hybridized with XAP-2 cDNA probe (B),
showing tissue specificity of KTF-l/XAP-2 DNA-binding
activity and RNA accumulation in stage 9 (late blastula)
and stage 19-24 (tailbud) Xenopus embryos. The amount
of whole cell extract per binding reaction, or RNA per
lane, corresponded to an equal number of cells (see
Materials and methods). The positions of the 2.6, 2.2 and
1.8 kb XAP-2 RNAs are indicated by arrows.
known to contain KTF-1 binding sites, the function of
KTF-l/XAP-2 in adult skin, kidney and brain must be
different from that in the embryonic epidermis (see
Discussion). Adult tissues expressed only the two
longer XAP-2 transcripts, lacking the 1.8 kb band,
which thus appears to be specific to the early embryo.
KTF-l/XAP-2 is present in mammalian keratinexpressing cells
The data illustrated in Fig. 5 show that a positive
transcriptional-regulatory element in the human keratin K14 gene can function as a KTF-1 and XAP-2
binding site, raising the question whether K14-transcribing mammalian cells specifically contain KTF-1
activity and AP-2 RNA. Mobility shift, PCBA, DNAbinding specificity and RNA gel blot experiments,
equivalent to those described above for Xenopus KTFl/XAP-2, were, therefore, carried out on cultured
mouse basal epidermal cells, which are strongly positive
for K14 expression (Yuspa et al. 1989; Vassar et al.
1989), and the human fibroblast cell line, WI-38 (K14
negative; Leask et al. 1990). Fig. 9A shows that whole
cell extract from the mouse basal cells gives a strong and
specific mobility shift with the standard X-157 probe,
while the WI-38 cells gave only a non-specific shift,
290
A. M. Snape, R. S. Winning and T. D. Sargent
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showing tissue specificity of KTF-l/XAP-2 DNA-binding activity and RNA accumulation in adult Xenopus tissues.
(A) DNA-mobility shift assay. For kidney and skin, competition analysis using unlabeled X-157 oligonucleotide, or X-157M,
as in Fig. 6B, shows that probe is shifted due to specific KTF-l/XAP-2 binding. (B) RNA gel blot. Each lane was loaded
with approximately the same amount of total RNA (4,ug) except for lung RNA, which was underloaded by about 50%. A
3 day exposure of the X-ray film is shown. After 3 weeks exposure no signal was seen in any of the negative lanes, except
for a barely detectable, diffuse band in lung, which may thus contain a very low level of XAP-2 RNA. The positions of the
2.6, 2.2 and 1.8 kb XAP-2 RNAs are indicated by arrows.
which was not competed by 1000-fold excess of
unlabeled probe. The basal-cell-derived complex
migrated with a mobility slightly faster than that of in
v/rro-synthesized XAP-2 (data not shown), as was also
observed for KTF-1 from tadpoles (Fig. 1). Figs 9C and
9D show that the KTF-l/AP-2 DNA-binding protein
from mouse cells is virtually indistinguishable, with
respect to its PCBA profile, and binding specificity,
from Xenopus KTF-l/XAP-2 (compare with Figs 2 and
5 and see Table 1). Moreover, mouse basal cells are
positive, and WI-38 cells negative, for AP-2 RNA
(Fig. 9B). These results suggest that tissue-specific
expression of KTF-l/AP-2 is conserved between
amphibians and mammals. The KTF-1 binding site at
-229 in the K14 promoter has already been demonstrated by Leask et al. (1990) to act as positive regulator
of transcription, and it seems likely that the protein that
binds this site, which they call KER-1, is KTF-l/AP-2,
or a close relative (see Discussion)
Discussion
KTF-1 and AP-2 are closely related or identical
The XK81A1 gene of Xenopus laevis encodes a type I
cytokeratin which is transcribed specifically in the
embryonic epidermis. Promoter binding studies using
crude embryonic extracts, combined with promoter
mutation analysis, led to the identification of an
activator of XK81A1 expression: KTF-1 (Keratin
Transcription Factor-1; Snape etal. 1990). In this paper,
further characterization of KTF-1 shows that it is either
closely related or identical to XAP-2, the Xenopus
homologue of the mammalian tissue-specific transcription factor AP-2.
To support this conclusion, we have presented
several lines of evidence, each of which is circumstantial
or correlative in nature, and none alone can be
considered conclusive. However, taken together these
experiments strongly support a close relationship
between these factors. Furthermore, our laboratory
isolated Xenopus AP-2 cDNA clones by reduced
stringency hybridization with a human AP-2 probe
(Winning et al. 1991). Several clones were isolated in
this way, and partial DNA sequence data revealed no
significant differences (L. Shea and S. Marcus, unpublished data). Therefore, if KTF-1 and AP-2 were not
encoded by the same gene, then one would need to
postulate two distinct proteins binding the same DNA
sequence element with the same affinity, exhibiting
similar or identical mobility in band shift experiments,
appearing in the same subset of tissues and at the same
time in development, sharing similar protease sensitivity and sharing two or more antigenic sites, but
nevertheless having insufficient sequence homology to
permit isolation of KTF-1 cDNA clones by hybridization with the human AP-2 probe. We regard this
proposition as highly improbable. However, our data
are consistent with the existence of subtle differences
between KTF-1 and XAP-2. The possibility of posttranslational modifications, alternative proteins generated from differently processed RNAs, or other minor
differences must be considered open.
,4P-2 and KTF-1 are related or identical
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Fig. 9. Analysis of KTF-l/AP-2 DNA-binding activity and AP-2 RNA accumulation in cultured mammalian cells.
(A) DNA-mobility shift assay with X-157 probe. For mouse basal epidermal whole cell extract, competition analysis using
unlabeled X-157, or X-157M, as in Fig. 6B, showed that probe was shifted due to specific KTF-l/AP-2 binding (closed
arrow"). Extract from WI-38 cells gave only a non-specific shift (open arrow), which was competed neither by X-157 nor by
X-157 . (B) RNA gel blot. Lanes were loaded with approximately 4,ug total RNA from mouse basal epidermal cells
(MBC) or WI-38 cells, and hybridized with a human AP-2 cDNA probe. (C) Proteolytic clipping bandshift assays of KTFl/AP-2 DNA-binding protein from mouse basal epidermal cells. Conditions were as in Fig. 2. Compare the patterns of
shifted bands with those in Fig. 2. In this experiment the gel electrophoresis was continued somewhat longer than in the
experiments shown in Fig. 2, and the unbound probe ran off the gel. Repeat assays of Xenopus tadpole extract KTF-1, and
in vitro translated XAP-2, run at the same time, gave equivalent patterns (not shown). (D) Bar charts illustrating DNAsequence binding specificity of KTF-l/AP-2 from mouse basal epidermal cells. Conditions were as in Fig. 5.
Developmental significance of the KTF-1 /XAP-2
relationship
We have previously shown (Snape et al. 1990) that the
major KTF-1 binding site at —157 is required for
efficient expression of the XK81A1 frog keratin gene,
supporting an important regulatory role for KTF-1 in
this context. Based on the results that we have
presented in this paper, this functional significance can
presumably be extended to XAP-2. The most obvious
mechanism for achieving tissue-specific gene expression
is by the localized activity of transcription factors.
Appropriately, in the tailbud stage embryo, KTF-1
DNA-binding activity is strikingly concentrated in the
epidermis, and this localization appears to be regulated,
at least in part, at the level of KTF-l/XAP-2 transcript
accumulation. XAP-2 transcripts were first detected
shortly after the MBT and, although the signal at this
stage was weak, this RNA appeared to be localized in
the animal hemisphere as early as the late blastula, with
enrichment in the epidermis persisting at the taibud
stage. However, there is evidence that KTF-l/XAP-2
activity may not be completely confined to the
epidermis; in earlier experiments, insertion of two
KTF-1 binding sites 500 bp upstream of a Xenopus
292
A. M. Snape, R. S. Winning and T. D. Sargent
/3-globin gene, which was then injected into embryos,
appeared to enhance transcription in both epidermal
and non-epidermal tissues (Snape et al. 1990). In part,
this is because globin RNA levels were measured on a
per embryo basis, whereas here KTF-l/XAP-2 was
evaluated on a per cell basis. Since the non-epidermal
fraction contains more cells per embryo than the
epidermis, this would exaggerate the level of globin
RNA in non-epidermal cells. In addition, although
KTF-l/XAP-2 transcripts were detected only in the
animal hemisphere of the blastula, this half of the
embryo contributes to other tissues besides epidermis,
and these non-epidermal tissues may transiently express
KTF-l/XAP-2, leading to enhanced expression of the
globin construct.
Transcripts from the XK81A1 gene are first detected
in the late blastula or early gastrula (stages 9 and 10),
peak in the tailbud embryo (stage 35/36), are greatly
reduced by stage 44, and are undetectable in the
recently metamorphosed frog (Miyatani et al. 1986; P.
Mathers, unpublished data). KTF-l/XAP-2 could
therefore be involved in establishing high level transcription of XK81A1, but KTF-1 DNA-binding activity
and XAP-2 RNA persist after the keratin gene is no
longer transcribed, suggesting that they also play a role
in the expression of other genes.
If KTF-l/XAP-2 can function as a transcriptional
activator in non-epidermal tissues, then it probably acts
in combination with other, positive and/or negative,
transcription factors to give completely epidermisspecific keratin expression. There is evidence, from in
vivo footprinting, for other factors binding the XK81A1
promoter (P. Mathers, unpublished data). It is
expected that these would play a role in tissue-specific
transcription, since mutation of the KTF-1 site at —157
leaves residual expression of keratin in the epidermis.
However, the keratin promoter used in these experiments retained the site at —254, which, although of
lower affinity than the —157 site, is shown here to bind
KTF-1 in vitro. Thus it is possible that the remaining
epidermal keratin transcription is still mediated by
KTF-1. Experiments to test this are currently in
progress.
KTF-l/XAP-2 is tissue specific in the adult frog
Since the XK81 embryonic keratins are not transcribed
in Xenopus after metamorphosis (except to a low level
in esophagus; Fouquet et al. 1988), the finding that
KTF-l/XAP-2 is expressed, in a highly tissue-specific
pattern, in adult skin, kidney and, to a lesser extent,
brain, suggests that, like other developmentally important genes, KTF-l/XAP-2 has an early embryonic
function and a separate phase of activity later in
development. Considering the high expression levels in
skin and kidney, it is tempting to speculate that KTFl/XAP-2 may play a role in the differentiation of adult
epithelia. In addition, AP-2 sites are important in the
expression of a variety of mammalian genes, including,
for example, tissue plasminogen activator (Rickles etal.
1989), growth hormone (Courtois et al. 1990) and glial
fibrillary acidic protein (Miura et al. 1990), so XAP-2
may regulate several different genes in Xenopus as well.
Mitchell et al. (1991) have shown recently that AP-2
RNA is localized in fetal and adult mouse tissues, and
shows a similar pattern of distribution to the one
described here in Xenopus, again suggesting conservation of AP-2 function between amphibians and
mammals.
A factor in mouse basal keratinocytes resembles KTF1/XAP-2
Of the mammalian keratins for which sequence is
published, human keratin K14 (Marchuk et al. 1985)
shows the greatest similarity to XK81A1 (58.5%
identity at the amino acid level). Sequence homology
extends outside the rod region into the globular
domains, which are usually less conserved, suggesting a
close evolutionary relationship for these genes. K14 is
specifically transcribed in the proliferating basal cell
layer of the adult skin, and is down-regulated as the
cells differentiate (Vassar et al. 1989; Tyner and Fuchs,
1986). The results presented in Fig. 9 strongly suggest
that there is a factor in mouse basal keratinocytes that is
very similar to KTF-1 and XAP-2. Leask et al. (1990)
mention unpublished results suggesting that KER-1, a
factor involved in regulating K14 gene expression in
human keratinocytes, can bind to the AP-2 site from the
human metallothionein gene. Presumably, the AP-2like mouse keratinocyte factor that we have identified
corresponds to KER-1.
In summary, our results support a major developmental role for transcription factor AP-2; the activation
of epidermal keratin gene expression in amphibian
embryos. The prospect for the study of embryonic
development is especially exciting, since it is by the
knowledge of factors regulating germ-layer-specific
gene expression, and an understanding of the mechanisms that regulate these factors, that we hope to work
back towards identification of the primary germ-layer
determinants. The epidermal cytokeratin gene is now
the earliest Xenopus germ-layer marker for which a
transcriptional activator is known, and the regulation of
KTF-l/XAP-2, since it is non-maternal and transcribed
specifically in the embryo, is itself accessible to further
study.
The authors would like to thank Dr T. Williams (U.C.
Berkeley) for making available the human AP-2 cDNA clone,
antisera, and unpublished data on human AP-2, Dr S. Yuspa
and Dr T. Howard (NIH) for cultured cells, and Dr V.
Agarwal (NIH) for RNA samples from adult Xenopus tissues.
We also thank Dr P. Mathers, L. Shea and S. Marcus for
unpublished data, and other members of the Sargent
laboratory for advice and encouragement. A.M.S. is supported in part by a grant from the Wellcome Trust. R.S.W. is
supported by a postdoctoral fellowship from the Natural
Sciences and Engineering Research Council of Canada.
References
AUSUBEL, F. M., BRENT, R., KINGSTON, R. E., MOORE, D. D . ,
SEIDMAN, J. G., SMITH, J. A. AND STRUHL, K. (1987). Current
Protocols in Molecular Biology, vol. 1. 2.7.1-2.7.5 and
3.5.7-3.5.10. New York: John Wiley and Sons.
288
A. M. Snape, R. S. Winning and T. D. Sargent
gene
A)
Xenopus keratins
position
sequence
competitor
name
AACAAACACCCTG AGGCTACGTA
TGCTGAAGGAAaCCTGaAGcaaGGAGAGAG
tqCCTGqqGcaq
tqCCTGqqGcaa
caCCTG AaGta
X-157
X-254
XK81A1
XK81A1
XK81B1
XK81B2
XK70A
-157
-254
-248
-240
-269
human keratin
K14
K14
-86
-229
human metallothionein
hMT-ll.
-175
GAACTGACcqCCcG cGGCcCGTGTGCAGAG
mutated sequences
XK81A1
XK81A1
hMT-llA
-157
-254
-175
AACAAACAtttca qaatTACGTA
TGCTGAAGGAAt at cGat t caaGGAGAGAG
GAACTGACctttca qaatcCGTGTGCAGAG
TGGCTTTCATCACCCac AGGCTAGCGCCAACT
GGGAAAGTGTAaCCTGcAGGCcCACACCTCCC
K14-86
K14-229
MT-175
B)
X-1571
X-254M
MT-1751
Fig. 4. (A) Sequences of putative KTF-l/(X)AP-2 binding sites. In XK81A1 the underlined bases make up an 11 bp
imperfect palindrome, around a central G at -157. The position of the other sites is given as the position of the base
equivalent to that G. Bases in bold type correspond to those making up an AP-2 site, as defined by Mitchell et al. (1991).
Within the binding site, bases in lower case differ from those at equivalent positions in the XK81A1 -157 site. In some
cases, a gap has been left in the sequence, to facilitate alignment of the sites. Where the site was used as a probe or
competitor in DNA-mobility shift assays, additional promoter sequence, outside the binding site, was included in the
oligonucleotide; these sequences are also given. References for these sequences are as follows: XK81A1, Bl and B2,
Miyatani et al. (1986); XK70A, Krasner et al. (1988) and GenBank accession no. M59455; K14, Marchuk et al. (1985);
hMT-IIA, Karin and Richards (1982). (B) Sequences used as mobility shift competitors, corresponding to promoter
sequences with bases altered so as to eliminate KTF-l/(X)AP-2 binding.
followed by an increase at around the feeding tadpole
stage (stage 45). Thus the developmental profiles of
KTF-1 protein and XAP-2 RNA are similar, consistent
with the XAP-2 gene encoding KTF-1.
It was also interesting to find out whether KTF-1
DNA-binding activity and accumulation of XAP-2
RNA corresponded with respect to tissue specificity.
Therefore, whole cell protein extracts and total RNA
were prepared from late-blastula-stage Xenopus embryos dissected into animal (pigmented) and vegetal
(non-pigmented) halves, tailbud-stage Xenopus embryos dissected into epidermal and non-epidermal
fractions, and a selection of adult Xenopus tissues. For
each source, RNA gel blots were probed for XAP-2
RNA, while mobility shift assays for KTF-1 were
carried out using whole cell extracts (Figs 7 and 8).
When assaying KTF-1 DNA-binding activity in
dissected embryos (Fig. 7A), the genomic DNA content of each fraction was compared (see Materials and
methods) and the volume of extract added to the DNAbinding reactions was modified accordingly, so that
each contained extract from an equivalent number of
cells. At the blastula stage there was enrichment of
KTF-1 in the animal half, from which the epidermis,
plus other tissues, derives and, by the tailbud stage,
KTF-1 activity was strikingly localized in the epidermal
A)
Competitors:
X-157
X-157 M
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Fig. 5. Bar charts illustrating DNA-sequence
binding specificity of KTF-1 from
metamorphosing tadpole extracts (A) and in
vitro translated XAP-2 (B). Unlabeled
oligonucleotides were used in DNA-mobility
shift assays as competitors for binding to the
32
P-labeled X-157 probe, at 5-, 50- and 500times the probe concentration. In each case,
the intensity of the shifted band was
compared to the intensity of the band shifted
when no oligonucleotide competitor was
added. Results are plotted on the vertical axis
as percentage competition, i.e. 100%
competition indicates that there was complete
elimination of the shifted band. Each result is
the mean of two experiments; the data for
experimental competitors are presented in
Table 1.
AP-2 and KTF-1 are related or identical
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Fig. 6. (A) DNA-mobility shift assay showing time course
of KTF-1 DNA-binding activity during Xenopus embryonic
development. The probe is X-157. Whole cell extract was
prepared from 100 embryos of each stage shown
(Nieuwkoop and Faber, 1967), and extract equivalent to •
that from 2.5 embryos was used in each reaction. The
lowest band represents unbound probe. The open arrow
indicates probe shifted by a non-specific binding reaction.
The closed arrow indicates probe shifted by specific KTF-1
binding. (B) Competition analysis showing non-specific
DNA-binding activity from stage 1 embryos (fertilized
eggs), not competed efficiently by oligonucleotide probes
(open arrow) versus specific KTF-1 activity (closed arrow)
from stage 10 embryos (gastrulae), competed by 1000-fold
excess of unlabeled probe (X-157) oligonucleotide, but not
by 1000-fold excess of X-157M, the equivalent sequence
with the KTF-1-binding site mutated.
fraction. When the RNA gel blot (Fig. 7B) was loaded
in the equivalent fashion (RNA from an equal number
of cells in each lane), XAP-2 transcripts were localized
in the blastula animal half, and the tailbud epidermis.
Once again, the evidence is consistent with the
hypothesis that XAP-2 is KTF-1.
In adult Xenopus tissues, both KTF-1 activity and
XAP-2 RNA show similar, highly tissue-specific,
expression patterns (Fig. 8). In the KTF-1 assay, the
volume of extract in the DNA-binding reactions was
modified so that equal amounts of total protein were
compared for each tissue. Since many of the extracts
appeared negative for KTF-1, the experiment was then
repeated with the maximum possible volume of extract
in each reaction. Still, KTF-1 activity could not be
detected in ovary, liver or lung. Adult skin and kidney,
however, both contained significant KTF-1 activity.
When total RNA was probed for XAP-2 RNA,
transcripts were seen in skin and kidney, but not in
oocyte, liver or lung RNA. This is further strong
evidence that KTF-1 and XAP-2 are the same factor. In
addition, testis RNA is negative and brain RNA is
weakly positive for XAP-2 transcripts. Protein extracts
were not prepared from these tissues, so there are no
data on their KTF-1 activity. Since adult Xenopus
tissues (except possibly the esophageal lining; Fouquet
et al. 1988) do not express the embryonic keratin genes
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RNA gel blot hybridized with XAP-2 cDNA probe (B),
showing tissue specificity of KTF-l/XAP-2 DNA-binding
activity and RNA accumulation in stage 9 (late blastula)
and stage 19-24 (tailbud) Xenopus embryos. The amount
of whole cell extract per binding reaction, or RNA per
lane, corresponded to an equal number of cells (see
Materials and methods). The positions of the 2.6, 2.2 and
1.8 kb XAP-2 RNAs are indicated by arrows.
known to contain KTF-1 binding sites, the function of
KTF-l/XAP-2 in adult skin, kidney and brain must be
different from that in the embryonic epidermis (see
Discussion). Adult tissues expressed only the two
longer XAP-2 transcripts, lacking the 1.8 kb band,
which thus appears to be specific to the early embryo.
KTF-l/XAP-2 is present in mammalian keratinexpressing cells
The data illustrated in Fig. 5 show that a positive
transcriptional-regulatory element in the human keratin K14 gene can function as a KTF-1 and XAP-2
binding site, raising the question whether K14-transcribing mammalian cells specifically contain KTF-1
activity and AP-2 RNA. Mobility shift, PCBA, DNAbinding specificity and RNA gel blot experiments,
equivalent to those described above for Xenopus KTFl/XAP-2, were, therefore, carried out on cultured
mouse basal epidermal cells, which are strongly positive
for K14 expression (Yuspa et al. 1989; Vassar et al.
1989), and the human fibroblast cell line, WI-38 (K14
negative; Leask et al. 1990). Fig. 9A shows that whole
cell extract from the mouse basal cells gives a strong and
specific mobility shift with the standard X-157 probe,
while the WI-38 cells gave only a non-specific shift.
290
A. M. Snape, R. S. Winning and T. D. Sargent
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showing tissue specificity of KTF-l/XAP-2 DNA-binding activity and RNA accumulation in adult Xenopus tissues.
(A) DNA-mobility shift assay. For kidney and skin, competition analysis using unlabeled X-157 oligonucleotide, or X-157M,
as in Fig. 6B, shows that probe is shifted due to specific KTF-l/XAP-2 binding. (B) RNA gel blot. Each lane was loaded
with approximately the same amount of total RNA (4,ug) except for lung RNA, which was underloaded by about 50%. A
3 day exposure of the X-ray film is shown. After 3 weeks exposure no signal was seen in any of the negative lanes, except
for a barely detectable, diffuse band in lung, which may thus contain a very low level of XAP-2 RNA. The positions of the
2.6, 2.2 and 1.8 kb XAP-2 RNAs are indicated by arrows.
which was not competed by 1000-fold excess of
unlabeled probe. The basal-cell-derived complex
migrated with a mobility slightly faster than that of in
v/rro-synthesized XAP-2 (data not shown), as was also
observed for KTF-1 from tadpoles (Fig. 1). Figs 9C and
9D show that the KTF-l/AP-2 DNA-binding protein
from mouse cells is virtually indistinguishable, with
respect to its PCBA profile, and binding specificity,
from Xenopus KTF-l/XAP-2 (compare with Figs 2 and
5 and see Table 1). Moreover, mouse basal cells are
positive, and WI-38 cells negative, for AP-2 RNA
(Fig. 9B). These results suggest that tissue-specific
expression of KTF-l/AP-2 is conserved between
amphibians and mammals. The KTF-1 binding site at
-229 in the K14 promoter has already been demonstrated by Leask et al. (1990) to act as positive regulator
of transcription, and it seems likely that the protein that
binds this site, which they call KER-1, is KTF-l/AP-2,
or a close relative (see Discussion)
Discussion
KTF-1 and AP-2 are closely related or identical
The XK81A1 gene of Xenopus laevis encodes a type I
cytokeratin which is transcribed specifically in the
embryonic epidermis. Promoter binding studies using
crude embryonic extracts, combined with promoter
mutation analysis, led to the identification of an
activator of XK81A1 expression: KTF-1 (Keratin
Transcription Factor-1; Snape etal. 1990). In this paper,
further characterization of KTF-1 shows that it is either
closely related or identical to XAP-2, the Xenopus
homologue of the mammalian tissue-specific transcription factor AP-2.
To support this conclusion, we have presented
several lines of evidence, each of which is circumstantial
or correlative in nature, and none alone can be
considered conclusive. However, taken together these
experiments strongly support a close relationship
between these factors. Furthermore, our laboratory
isolated Xenopus AP-2 cDNA clones by reduced
stringency hybridization with a human AP-2 probe
(Winning et al. 1991). Several clones were isolated in
this way, and partial DNA sequence data revealed no
significant differences (L. Shea and S. Marcus, unpublished data). Therefore, if KTF-1 and AP-2 were not
encoded by the same gene, then one would need to
postulate two distinct proteins binding the same DNA
sequence element with the same affinity, exhibiting
similar or identical mobility in band shift experiments,
appearing in the same subset of tissues and at the same
time in development, sharing similar protease sensitivity and sharing two or more antigenic sites, but
nevertheless having insufficient sequence homology to
permit isolation of KTF-1 cDNA clones by hybridization with the human AP-2 probe. We regard this
proposition as highly improbable. However, our data
are consistent with the existence of subtle differences
between KTF-1 and XAP-2. The possibility of posttranslational modifications, alternative proteins generated from differently processed RNAs, or other minor
differences must be considered open.
,4P-2 and KTF-1 are related or identical
fk.
Extract:
Competitor:
Competitors:
B.
Mouse
Basal
Cells
X-157
c m
O
Z
in
X-157
0*
WI-38
£
in
Mouse Basal Cell Extract
Endoproteinase
ArgC
in
z
X-254
291
X-254 M
K14-229
K14-86
MT-175
_
Tr
.
VPsm
Endoproteinase
Glu C
MT-175*
o
a.
o
fl.
Fold Excess Competitor
Fig. 9. Analysis of KTF-l/AP-2 DNA-binding activity and AP-2 RNA accumulation in cultured mammalian cells.
(A) DNA-mobility shift assay with X-157 probe. For mouse basal epidermal whole cell extract, competition analysis using
unlabeled X-157, or X-157M, as in Fig. 6B, showed that probe was shifted due to specific KTF-l/AP-2 binding (closed
arrowV Extract from WI-38 cells gave only a non-specific shift (open arrow), which was competed neither by X-157 nor by
X-157 . (B) RNA gel blot. Lanes were loaded with approximately 4,ug total RNA from mouse basal epidermal cells
(MBC) or WI-38 cells, and hybridized with a human AP-2 cDNA probe. (C) Proteolytic clipping bandshift assays of KTFl/AP-2 DNA-binding protein from mouse basal epidermal cells. Conditions were as in Fig. 2. Compare the patterns of
shifted bands with those in Fig. 2. In this experiment the gel electrophoresis was continued somewhat longer than in the
experiments shown in Fig. 2, and the unbound probe ran off the gel. Repeat assays of Xenopus tadpole extract KTF-1, and
in vitro translated XAP-2, run at the same time, gave equivalent patterns (not shown). (D) Bar charts illustrating DNAsequence binding specificity of KTF-l/AP-2 from mouse basal epidermal cells. Conditions were as in Fig. 5.
Developmental significance of the KTF-1 /XAP-2
relationship
We have previously shown (Snape et al. 1990) that the
major KTF-1 binding site at —157 is required for
efficient expression of the XK81A1 frog keratin gene,
supporting an important regulatory role for KTF-1 in
this context. Based on the results that we have
presented in this paper, this functional significance can
presumably be extended to XAP-2. The most obvious
mechanism for achieving tissue-specific gene expression
is by the localized activity of transcription factors.
Appropriately, in the tailbud stage embryo, KTF-1
DNA-binding activity is strikingly concentrated in the
epidermis, and this localization appears to be regulated,
at least in part, at the level of KTF-l/XAP-2 transcript
accumulation. XAP-2 transcripts were first detected
shortly after the MBT and, although the signal at this
stage was weak, this RNA appeared to be localized in
the animal hemisphere as early as the late blastula, with
enrichment in the epidermis persisting at the taibud
stage. However, there is evidence that KTF-l/XAP-2
activity may not be completely confined to the
epidermis; in earlier experiments, insertion of two
KTF-1 binding sites 500 bp upstream of a Xenopus
292
A. M. Snape, R. S. Winning and T. D. Sargent
/3-globin gene, which was then injected into embryos,
appeared to enhance transcription in both epidermal
and non-epidermal tissues (Snape et al. 1990). In part,
this is because globin RNA levels were measured on a
per embryo basis, whereas here KTF-l/XAP-2 was
evaluated on a per cell basis. Since the non-epidermal
fraction contains more cells per embryo than the
epidermis, this would exaggerate the level of globin
RNA in non-epidermal cells. In addition, although
KTF-l/XAP-2 transcripts were detected only in the
animal hemisphere of the blastula, this half of the
embryo contributes to other tissues besides epidermis,
and these non-epidermal tissues may transiently express
KTF-l/XAP-2, leading to enhanced expression of the
globin construct.
Transcripts from the XK81A1 gene are first detected
in the late blastula or early gastrula (stages 9 and 10),
peak in the tailbud embryo (stage 35/36), are greatly
reduced by stage 44, and are undetectable in the
recently metamorphosed frog (Miyatani et al. 1986; P.
Mathers, unpublished data). KTF-l/XAP-2 could
therefore be involved in establishing high level transcription of XK81A1, but KTF-1 DNA-binding activity
and XAP-2 RNA persist after the keratin gene is no
longer transcribed, suggesting that they also play a role
in the expression of other genes.
If KTF-l/XAP-2 can function as a transcriptional
activator in non-epidermal tissues, then it probably acts
in combination with other, positive and/or negative,
transcription factors to give completely epidermisspecific keratin expression. There is evidence, from in
vivo footprinting, for other factors binding the XK81A1
promoter (P. Mathers, unpublished data). It is
expected that these would play a role in tissue-specific
transcription, since mutation of the KTF-1 site at —157
leaves residual expression of keratin in the epidermis.
However, the keratin promoter used in these experiments retained the site at -254, which, although of
lower affinity than the —157 site, is shown here to bind
KTF-1 in vitro. Thus it is possible that the remaining
epidermal keratin transcription is still mediated by
KTF-1. Experiments to test this are currently in
progress.
KTF-l/XAP-2 is tissue specific in the adult frog
Since the XK81 embryonic keratins are not transcribed
in Xenopus after metamorphosis (except to a low level
in esophagus; Fouquet et al. 1988), the finding that
KTF-l/XAP-2 is expressed, in a highly tissue-specific
pattern, in adult skin, kidney and, to a lesser extent,
brain, suggests that, like other developmentally important genes, KTF-l/XAP-2 has an early embryonic
function and a separate phase of activity later in
development. Considering the high expression levels in
skin and kidney, it is tempting to speculate that KTFl/XAP-2 may play a role in the differentiation of adult
epithelia. In addition, AP-2 sites are important in the
expression of a variety of mammalian genes, including,
for example, tissue plasminogen activator (Rickles etal.
1989), growth hormone (Courtois et al. 1990) and glial
fibrillary acidic protein (Miura et al. 1990), so XAP-2
may regulate several different genes in Xenopus as well.
Mitchell et al. (1991) have shown recently that AP-2
RNA is localized in fetal and adult mouse tissues, and
shows a similar pattern of distribution to the one
described here in Xenopus, again suggesting conservation of AP-2 function between amphibians and
mammals.
A factor in mouse basal keratinocytes resembles KTF1/XAP-2
Of the mammalian keratins for which sequence is
published, human keratin K14 (Marchuk et al. 1985)
shows the greatest similarity to XK81A1 (58.5%
identity at the amino acid level). Sequence homology
extends outside the rod region into the globular
domains, which are usually less conserved, suggesting a
close evolutionary relationship for these genes. K14 is
specifically transcribed in the proliferating basal cell
layer of the adult skin, and is down-regulated as the
cells differentiate (Vassar et al. 1989; Tyner and Fuchs,
1986). The results presented in Fig. 9 strongly suggest
that there is a factor in mouse basal keratinocytes that is
very similar to KTF-1 and XAP-2. Leask et al. (1990)
mention unpublished results suggesting that KER-1, a
factor involved in regulating K14 gene expression in
human keratinocytes, can bind to the AP-2 site from the
human metallothionein gene. Presumably, the AP-2like mouse keratinocyte factor that we have identified
corresponds to KER-1.
In summary, our results support a major developmental role for transcription factor AP-2; the activation
of epidermal keratin gene expression in amphibian
embryos. The prospect for the study of embryonic
development is especially exciting, since it is by the
knowledge of factors regulating germ-layer-specific
gene expression, and an understanding of the mechanisms that regulate these factors, that we hope to work
back towards identification of the primary germ-layer
determinants. The epidermal cytokeratin gene is now
the earliest Xenopus germ-layer marker for which a
transcriptional activator is known, and the regulation of
KTF-l/XAP-2, since it is non-maternal and transcribed
specifically in the embryo, is itself accessible to further
study.
The authors would like to thank Dr T. Williams (U.C.
Berkeley) for making available the human AP-2 cDNA clone,
antisera, and unpublished data on human AP-2, Dr S. Yuspa
and Dr T. Howard (NIH) for cultured cells, and Dr V.
Agarwal (NIH) for RNA samples from adult Xenopus tissues.
We also thank Dr P. Mathers, L. Shea and S. Marcus for
unpublished data, and other members of the Sargent
laboratory for advice and encouragement. A.M.S. is supported in part by a grant from the Wellcome Trust. R.S.W. is
supported by a postdoctoral fellowship from the Natural
Sciences and Engineering Research Council of Canada.
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{Accepted 5 June 1991)