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Molecular and Cellular Biology, January 2000, p. 661-671, Vol. 20, No. 2
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel PF/PN Motif Inhibits Nuclear Localization
and DNA Binding Activity of the ESX1 Homeoprotein
Yu-Ting
Yan,1,2
Stacey M.
Stein,1,3
Jixiang
Ding,1,2
Michael M.
Shen,1,2,* and
Cory
Abate-Shen1,3,*
Center for Advanced Biotechnology and
Medicine1 and Departments of
Pediatrics2 and Neuroscience and Cell
Biology,3 UMDNJ-Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854
Received 12 July 1999/Returned for modification 19 August
1999/Accepted 27 September 1999
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ABSTRACT |
Despite their significance for mammalian embryogenesis, the
molecular mechanisms that regulate placental growth and development have not been well defined. The Esx1 homeobox gene is of
particular interest because it is among the few regulatory genes that
have specific expression and function in the placenta during murine development. In addition, the ESX1 protein contains several notable features that are not often associated with homeoproteins, including an
atypical homeodomain of the paired-like class, a
proline-rich region that contains an SH3 binding motif, and a novel
repeat region consisting of prolines alternating with phenylalanines or
asparagines that we term the PF/PN motif. We have found that the ESX1
protein is expressed in the labyrinth layer of the placenta in vivo,
where its subcellular localization is primarily cytoplasmic. Our
results suggest that this unexpected subcellular localization is
conferred by the PF/PN motif, which inhibits nuclear localization of
ESX1 in cell culture, as well as its DNA binding activity in vitro.
Finally, we show that the proline-rich region of ESX1 mediates interactions in vitro with the c-abl SH3 domain as well as
with certain WW domains. We propose that the PF/PN motif provides a novel mechanism for regulating nuclear entry and that the essential function of ESX1 during placental development is mediated by its ability to couple cytoplasmic signal transduction events with transcriptional regulation in the nucleus.
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INTRODUCTION |
During mammalian embryogenesis, the
placenta provides a physical connection between the embryo and the
mother, supplying nutrients that are critical for embryonic growth and
survival. The multiple steps involved in the formation of the
chorioallantoic placenta are poorly understood at the molecular level,
yet they represent fundamental aspects of embryogenesis that are
essential for development to term (13). Indeed, defects of
implantation and placental development account for approximately
one-third of spontaneous abortions in humans, and even minor defects in
placentation can have severe consequences for embryo viability
(reviewed in reference 15).
Among the candidate regulatory genes that have been implicated in
placental development are the divergent homeobox gene Pem (28, 52), the zinc finger gene Rex-1
(40), the basic helix-loop-helix transcription factor
Mash-2 (19), and the nuclear hormone receptor ERR-
(29, 34). In addition, we and others have
been investigating Esx1 (also known as Spx1),
which is a murine homeobox gene of the "paired-like"
class (5, 26; Y.-T. Yan, S. Stein, P. Sciavolino, L. Yang, H. Wang, C. Abate-Shen, and M. M. Shen, unpublished
observations). Esx1 is an X-linked gene that is
chromosomally imprinted and is expressed specifically in extraembryonic
tissues during development, as well as in the adult testis (5, 25,
26). Consistent with its restricted expression pattern, loss of
function of Esx1 through targeted gene disruption leads to
overgrowth and defective morphogenesis of the labyrinth layer of the
placenta (25). These defects are consistent with an
essential role for Esx1 in establishing and/or maintaining
the maternal-fetal interface.
In addition to its restricted expression and function in extraembryonic
tissues, the Esx1 homeobox gene is of particular interest because its predicted protein sequence includes several motifs that are
generally not associated with homeoproteins. In this study, we have
examined the expression pattern of Esx1 transcripts and
corresponding protein product during placental development in the
mouse. Unexpectedly, we found that ESX1 protein was localized primarily
to the cytoplasm in vivo and in transfected cells and that a novel
motif that we termed the PF/PN domain inhibited its nuclear
localization. Furthermore, we showed that ESX1 contains a proline-rich
SH3 binding motif that mediates interactions with the c-abl
SH3 domain in vitro. These findings raise the possibility that ESX1
links cytoplasmic signaling events with nuclear transcriptional regulation during extraembryonic development.
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MATERIALS AND METHODS |
ES cell culture and cDNA library screening.
Culture of D3 ES
cells (16) and differentiation of embryoid bodies were
carried out as previously described (43, 44). To identify
novel paired-like homeodomains, we designed an 1,152-fold degenerate oligonucleotide 5'
C(G/T)(G/C/T)C(G/T)(A/G)TT(C/T)T(T/G)(G/A)AACCA(G/C/A)AC(T/C)TG 3' as a
probe to recognize the amino acid sequence QVWF(Q/K)NRR. Screening of a
primary cDNA library constructed from embryoid bodies on day 5 of
differentiation was performed in the presence of 3 M
tetramethylammonium chloride (Aldrich) (8). Screening of
200,000 primary phages resulted in the identification of a single
homeobox-containing clone that corresponds to Esx1. We subsequently identified six additional Esx1 clones through
rescreening of the embryoid-body cDNA library with a 5' fragment of the
original clone (see Fig. 1B, probe 2).
Analysis of Esx1 mRNA and protein expression.
Intact embryos and extraembryonic membranes were dissected on days 7.5 through 12.5 post coitum, where 0.5 day post coitum is defined as noon
of the day of the vaginal plug. RNase protection assays were carried
out as described previously with total RNA prepared from embryonic or
adult tissues (43). Nonradioactive in situ hybridization
with digoxygenin-labeled riboprobes was performed on whole mounts or
cryosections of embryos and extraembryonic tissues as described
previously (42, 44). No specific staining was detected when
a corresponding sense riboprobe for Esx1 was used (data not shown).
To generate polyclonal anti-ESX1 antisera, the homeodomain region of
ESX1 (amino acids 183 to 248) was subcloned into plasmid pQE-9 (Qiagen) and the resulting hexahistidine fusion
protein was purified by nickel affinity chromatography as described
previously (9). The recombinant ESX1(183-248) protein was
used as antigen to generate antisera in rabbits (Cocalico Biologicals).
Affinity-purified immunoglobulin G (IgG) was prepared by chromatography
of the anti-ESX1 antisera on an affinity resin in which the purified
ESX1 antigen had been covalently cross-linked to CNBr-activated
Sepharose (Pharmacia). For immunohistochemistry, cryosections (12 µ)
were fixed in 4% paraformaldehyde and blocked with 3% hydrogen
peroxide in methanol and normal goat serum. The sections were then
incubated with affinity-purified anti-ESX1 IgG (1:100) followed by
biotin-conjugated secondary antibody (1:200) and solutions A and B
provided with the VectaStain Elite ABC kit (Vector Laboratories).
Staining was visualized with 3,3'-diaminobenzidine tetrahydrochloride
enhanced with nickel chloride (Vector Laboratories) and was followed by
counterstaining with nuclear fast red (Vector Laboratories). No
specific staining was detected following blocking of anti-ESX1 IgG by
preincubation with purified ESX1 protein or in the absence of anti-ESX1
IgG (data not shown).
Production of ESX1 proteins and expression in mammalian
cells.
Sequences corresponding to the full-length ESX1 coding
region or various truncated derivatives were isolated by PCR
amplification from a cDNA corresponding to transcript A (see Fig. 1),
using primers containing EcoRI and XhoI
restriction sites to facilitate cloning into pcDNA3 (Invitrogen). The
5' PCR primers also contained sequences encoding an initiator
methionine and a single Myc epitope, to ensure uniformity at the 5'
end. To produce ESX1 proteins in mammalian cells, these pcDNA3-Esx1
plasmids were transiently transfected into COS-1 cells. For Western
blot analysis, transfected COS-1 cells were lysed in sodium dodecyl
sulfate (SDS) sample buffer, cell lysates were resolved by
SDS-polyacrylamide gel electrophoresis (PAGE) and proteins were
detected with anti-ESX1 antisera (1:5,000), followed by
chemiluminescence detection (Amersham). For indirect immunofluorescence, transfected COS-1 cells were fixed in 3%
paraformaldehyde and incubated with anti-ESX1 antisera (1:300). The
cells were then incubated in fluorescein-conjugated goat anti-rabbit
IgG (1:200) (Vector Laboratories) and subjected to nuclear staining with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; 30 nM). Antibody specificity was verified in control experiments with pre-immune IgG or secondary antisera alone.
In vitro DNA binding assays and GST interaction assays.
Electrophoretic mobility shift assays were performed as described
previously (9) using proteins produced by in vitro
transcription and translation from the pcDNA3-Esx1 plasmids. The DNA
sites were (top strand shown) 5'-ACACTAATTGGAGGC-3' (site 6 of reference 9), 5'-ACAATAATTGGAGGC-3'
(site 6-5 of reference 9),
5'-ACACTAATGGAGGC-3' (site 6-11 of reference
9), 5'-ACACTACTTGGAGGC-3' (site 6-19 of
reference 9), 5'-GATAATTGATTATC-3' (P2
site of reference 53), and
5'-ACTAATTGAATTAGC-3' (PRDQ9 site of reference
45).
For protein interaction assays, 11-mer peptide sequences from the ESX1
proline-rich domain (E#1, E#2, and E#3 [see Fig.
1A]),
the SH3
domains from c-
abl, c-
fyn, and N-
src
(
10), and the WW
domains of formin-binding protein 11 (FBP-11WW) and formin-binding
protein 30 (FBP-30WW) (
3) were
cloned into pGEX-2TK for production
of glutathione
S-transferase (GST) fusion proteins (
23).
Far-Western
protein interaction assays were performed as described
previously
(
10) with
32P-labeled GST-SH3 domain
fusion proteins to probe total-cell lysates
containing the GST ESX1
fusion peptides (GST E#1, GST E#2, and
GST E#3). GST interaction assays
were performed as described previously
(
55) with
35S-labeled ESX1 proteins obtained by in vitro
transcription-translation
and the purified GST-SH3 or GST-WW domain
fusion
proteins.
Nucleotide sequence accession numbers.
Transcripts A and B
have been deposited in GenBank under accession no. AF017735 and
AF017734, respectively.
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RESULTS |
Two distinct Esx1 transcripts have differing expression
patterns.
We have previously used the differentiation of embryonic
stem (ES) cells in culture as a model system for identifying genes involved in embryonic and extraembryonic development (43,
44). ES cells are pluripotent progenitors that spontaneously form
cell aggregates termed embryoid bodies in suspension culture. These embryoid bodies differentiate into a wide range of embryonic
derivatives, as well as extraembryonic derivatives such as visceral and
parietal extraembryonic endoderm (16).
To identify novel homeobox genes expressed during ES cell
differentiation, we screened a cDNA library constructed on day 5
of
embryoid-body culture, which is prior to the appearance of
many markers
of terminal differentiation (
43). Our screen was
focused on
isolating members of the
paired-like class of homeobox
genes, which are relatively underrepresented in sequence databases
(
7). Members of this class of homeobox genes display overall
similarity to the Drosophila
paired homeobox, but
unlike prototypic
paired/Pax genes, they lack sequences
encoding a second DNA binding
region (the
paired domain). In
addition,
paired-like homeobox
genes encode a glutamine at
position 50 of the homeodomain instead
of the serine characteristic of
paired/Pax homeobox genes (
7),
which is notable
since the residue at position 50 is an important
determinant of DNA
binding specificity (
20,
50). Consequently,
we designed a
degenerate oligonucleotide to recognize the sequence
QVWF(Q/K)NRR,
which is representative of many members of the
paired-like homeodomain subclass. Among the genes isolated by this approach
was the
paired-like homeobox gene
Esx1 (
5,
26).
Sequence analysis of seven independent
Esx1 cDNA clones
revealed the presence of two distinct transcripts that differ at their
5' ends (transcripts A and B [Fig.
1A
and B]). We examined the
expression of these two
Esx1
transcripts by RNase protection analysis
with an antisense probe that
produces two protected bands corresponding
to transcripts A and B (Fig.
1B and C). We found that
Esx1 expression
was limited to
extraembryonic tissues and to the adult testis
(Fig.
1C), consistent
with previous reports (
5,
26). Furthermore,
the relative
abundance of the two
Esx1 transcripts varies between
different tissues, with transcript A being more abundant in the
testis,
transcript B being more abundant in the placenta, and
equivalent levels
being found in undifferentiated ES cells (Fig.
1C).
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FIG. 1.
Differential expression of alternative Esx1
transcripts in the placenta and testes. (A) Amino acid sequence encoded
by the longest Esx1 transcript (transcript A)
(5). The putative initiator methionines for the products of
transcripts A and B are boxed (transcript B) (26). Protein
domains are indicated by boxes, the proline-rich peptides (E#1, E#2,
and E#3) are bracketed, potential phosphorylation sites in the
N-terminal extension are underlined, and the proline at position 43 of
the homeodomain is indicated by an arrow. (B) Schematic diagram of the
protein products of transcripts A and B. The protein regions depicted
are the N-terminal extension, the glutamic acid- and glutamine-rich
region (E/Q-rich), the homeodomain, the proline-rich region (Pro-rich),
and the PF/PN motif. The arrow indicates the position in transcript B
where its nucleotide sequence diverges at the 5' end from transcript A. Probes for in situ hybridization (probe 1) and RNase protection assays
(probe 2) are indicated. (C) RNase protection analysis of
Esx1 transcripts. Each hybridization mixture contained 15 µg of total RNA (or 50 µg of yeast tRNA) and antisense riboprobes
for Esx1 (probe 2) and ribosomal protein L32
(rpL32) as an internal standard (43). The
positions of protected fragments corresponding to transcripts A and B
are indicated.
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Because their 5' sequences are distinct, the protein product of
transcript B is predicted to begin at an internal methionine
and to
lack 68 residues at the N terminus of the product of transcript
A (Fig.
1A and B). Aside from their differing N-terminal regions,
the products
of transcripts A and B are otherwise identical and
contain several
notable features: (i) a divergent
paired-like homeodomain;
(ii) a proline-rich region that contains a putative
SH3 binding motif;
(iii) a region consisting almost exclusively
of glutamic acids and
glutamines (E/Q-rich region), which may
correspond to a
transcriptional activation domain; and (iv) an
unusual repeat sequence
consisting of prolines alternating with
phenylalanines or asparagines,
that we termed the PF/PN motif,
which is not found in known proteins in
sequence databases (Fig.
1A and B). Therefore, the predicted ESX1
protein displays a complex
modular organization, since alternative
transcripts encode proteins
with distinct N-terminal regions that have
features of transcription
factors (the homeodomain and glutamic
acid/glutamine-rich regions)
and signaling molecules (putative SH3
binding domains), as well
as other novel regions (the PF/PN
motif).
The subcellular distribution of ESX1 protein is primarily
cytoplasmic in the developing placenta.
A notable feature of the
Esx1 expression pattern during development is that it is
restricted to extraembryonic tissues, as shown by published in situ
hybridization studies (26) and by our RNase protection
analysis (Fig. 1C). We now show that Esx1 is specifically
expressed during early development in extraembryonic tissues that form
the placenta and subsequently continues to be expressed in the
labyrinth layer of the placenta, where its protein product is localized
primarily to the cytoplasm (Fig. 2 and
3).
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FIG. 2.
Esx1 expression in extraembryonic tissues. (A
and B) Schematic depiction of stages in extraembryonic development
using nomenclature from a previous report (24). (C to E)
Whole-mount in situ hybridization analysis of intact egg cylinders. (C)
Expression of Esx1 in the chorion (arrow) on day 8.0 (2 somites), just prior to fusion with the allantois (arrowhead). (D)
Punctate staining in the developing placenta (arrow) on day 8.5, shortly after chorioallantoic fusion. (E) View of the day 8.5 placenta
from below with the embryo removed; the ectoplacental cone and visceral
yolk sac are beneath the plane of focus. Esx1 expression
appears highest around the edges of the placenta (arrow). (F to K)
Section in situ hybridization to cryosections of egg cylinders on days
7.5 through 9.5 of gestation. (F) Sagittal section through a day 7.5 egg cylinder (head-fold stage); the ectoplacental cone is to the right.
Esx1 is expressed in the chorion (arrow) but not in the
visceral yolk sac (arrowhead). (G) Higher-power view of panel F,
showing staining of the chorionic ectoderm (arrows) but not of the
chorionic mesoderm (arrowhead). (H) Sagittal section on day 8.5 of
gestation, with scattered Esx1 expression in the newly
formed placenta, with the highest levels at the inverted lateral
margins (laminae) of the ectoplacental plate (arrows); no expression is
observed in the allantois (arrowhead). (I) Higher-power view of the
ectoplacental plate from panel H. (J) Low-power view of the placenta on
day 9.5 of gestation. Expression is found in the developing labyrinth
layer (arrow) but not in the spongiotrophoblast layer (arrowhead).
Expression is also evident in the visceral endoderm of the yolk sac
(small arrow); note that this is consistent with the expression of
Esx1 during differentiation of ES embryoid bodies, which
produce visceral endoderm. (K) Higher-power view of the labyrinth
layer. In panels C to E, bars represent 200 µm; in panels F to K,
bars represent 50 µm.
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FIG. 3.
ESX1 protein is localized primarily to the cytoplasm of
extraembryonic tissues. (A to C) Section in situ hybridization analysis
of Esx1 expression in the placenta on days 10.5, 11.5, and
12.5 of gestation. Arrows point to intense punctate expression at the
edge of the labyrinth layer of the placenta on days 10.5 and 11.5; note
the lack of expression in the adjacent allantoic plate (arrowheads). (D
to I) Immunohistochemical staining of ESX1 protein expression in the
placenta on days 10.5, 11.5, and 12.5. Note that the darker staining
(brown) corresponds to ESX1 immunoreactivity whereas the light color
(pink) corresponds to the counterstain with nuclear fast red. (D to F)
Low-power views of ESX1 protein expression in the labyrinth layer of
the placenta; note the overall similarity to the in situ hybridization
results. As in panels A to C, arrows indicate expression at the edge of
the labyrinth layer and arrowheads indicate the adjacent allantoic
plate. (G to I) High-power views show intense ESX1 expression in a
subpopulation of the labyrinth cells on day 10.5 (G) and on day 11.5 (H
and I), with localization predominantly to the cytoplasm (arrows) and
with scattered nuclear staining (arrowheads). Bars, 25 µm.
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In the mouse, the placenta develops from a complex association of
tissue layers that are derived from several distinct extraembryonic
lineages, as shown in Fig.
2A and B, which schematically display
these
tissue layers prior to and following placenta formation.
Unlike other
tissues, the placenta is derived from both the trophectoderm
and the
inner cell mass of the blastocyst (reviewed in reference
15). The trophectoderm gives rise to the chorionic
ectoderm,
which will contribute to the labyrinth layer of the placenta,
and it also gives rise to the ectoplacental cone, which ultimately
forms the trophoblast giant cells and spongiotrophoblast layers
of the
placenta. The inner cell mass, which also gives rise to
the embryo
proper, generates extraembryonic mesoderm and the allantois,
as well as
the extraembryonic parietal and viceral endoderm. Placenta
formation
occurs around embryonic day 8.5 when the allantois fuses
with the
chorion, with the chorionic tissue subsequently forming
the labyrinth
layer of the placenta (
24,
33). Chorioallantoic
fusion
allows nutrient exchange between the mother and the fetus,
with the
labyrinth layer providing the primary maternal-fetal
interface.
To examine the distribution of
Esx1 transcripts during
development, we performed in situ hybridization analysis from days
7.5 to 12.5 of mouse gestation by using digoxygenin-labeled riboprobes,
which afford a high degree of cellular and anatomical resolution.
Our
in situ analysis, which confirms and extends previous studies
(
26), shows that
Esx1 was expressed during late
gastrulation
(on day 7.75) in the chorionic ectoderm, prior to its
fusion with
the allantois (Fig.
2C and F). At this stage, expression
was undetectable
in any other extraembryonic tissue, including the
ectoplacental
cone and the visceral endoderm (Fig.
2C, F, and G).
Following
chorioallantoic fusion,
Esx1 was expressed in the
ectoplacental
plate (on day 8.5) and the developing labyrinth layer of
the placenta
(on day 9.5) (Fig.
2D, E, and H to K). Interestingly,
Esx1 was
expressed in a punctate fashion within the
ectoplacental plate
and was most highly expressed in its inverted
lateral margins
(laminae), as is evident by both whole-mount and
section in situ
hybridization (Fig.
2E and H).
Esx1
expression continued in the
labyrinth layer of the placenta from day
9.5 onward, with a peak
of expression on days 10.5 and 11.5 but with
decreased expression
on day 12.5 (Fig.
2J and K; Fig.
3A to
C).
To investigate the distribution of ESX1 protein, we performed
immunohistochemical analysis of placental tissues by using
affinity-purified
anti-ESX1 IgG (Fig.
3D to I). We found that the
distribution of
ESX1 protein in the placenta mirrored that of its
corresponding
mRNA (compare Fig.
3A to C with Fig.
3D to F). Thus,
anti-ESX1
staining is restricted to the labyrinth layer of the
placenta,
where it is concentrated in the outer margin and appears to
have
a punctate distribution. The anti-ESX1 staining was detectable
in
the placenta on day 9.5, becoming most intense on days 10.5
and 11.5 and decreasing in intensity by day 12.5 (Fig.
3D to F)
(data not
shown), which is similar to the expression of
Esx1
transcripts.
Notably, the peak expression of
Esx1
transcripts and protein occurring
on day 11.5 corresponds temporally to
the initial appearance of
the placental defects in
Esx1
knockout mice (
25).
High-power views revealed an unexpected feature of the anti-ESX1
staining pattern, namely, that it was primarily cytoplasmic
in a
majority of the immunostained cells (Fig.
3G to H). In contrast,
only a
subset of the cells displayed the nuclear staining that
would be
anticipated for a putative transcription factor. We have
also observed
a cytoplasmic distribution of anti-ESX1 staining
in the adult testis
(data not shown), another major site of
Esx1 expression
(Fig.
1C) (
5). The observed cytoplasmic localization
of ESX1
protein has interesting implications for its potential
function and/or
mode of regulation during placental
development.
The PF/PN motif confers cytoplasmic localization of ESX1.
To
investigate the contribution of the various protein regions of ESX1 to
its subcellular localization and other biochemical activities, we
produced a series of truncated ESX1 proteins, which contained the
homeodomain but lacked different portions of the N- or C-terminal
regions (Fig. 4A). Since ESX1 has two
alternative transcripts that differ at their 5' ends (Fig. 1A and B),
we designated the putative initiator methionine of the longer form
(transcript A) amino acid 1 [ESX1(1-382)] and that of the shorter
form (transcript B) as amino acid 69 [ESX1(69-382)] (Fig. 1A,
boxed). Each of the truncated ESX1 proteins was stably expressed
following in vitro translation or transient transfection in mammalian
cells (Fig. 4B).
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FIG. 4.
Analysis of ESX1 protein motifs. (A) Diagram of the
full-length and truncated ESX1 proteins. Shown are the glutamic acid-
and glutamine-rich region (E/Q), the homeodomain (HD), the proline-rich
region (Pro), and the PF/PN region. The numbering of amino acid
sequences (indicated in parentheses) is based on the product of
transcript A, such that ESX1(1-382) corresponds to the product of
transcript A and ESX1(69-382) corresponds to the product of transcript
B. Also indicated are the positions of the two alternative initiator
methionines for transcripts A and B at amino acids 1 and 69, respectively. The truncated ESX1 proteins also contain an N-terminal
Myc epitope as indicated. The table on the right summarizes the
subcellular localization, DNA binding properties, and SH3/WW domain
interactions for each ESX1 protein; primary data are shown in Fig. 5 to
7. Symbols: +++, strong DNA binding activity; ++ and +, weaker binding
activity;  , no detectable binding activity; ND, not determined. (B)
Comparison of ESX1 proteins produced by in vitro
transcription-translation or by transfected mammalian cells. For in
vitro transcription-translation (left), pcDNA3-Esx1 plasmids encoding
the indicated proteins were used to synthesize the corresponding mRNA
and this was followed by translation with rabbit reticulyocyte lysate
in the presence of [35S]methionine. In vitro-translated
proteins (3 µl) were resolved by SDS-PAGE (12% polyacrylamide) and
visualized by autoradiography. For expression in mammalian cells
(right), these pcDNA3-Esx1 plasmids were transfected into COS1 cells
and cellular proteins were extracted in SDS lysis buffer. Cell lysates
(15 µl) were resolved by SDS-PAGE (12% polyacrylamide) and
visualized by Western blot analysis with anti-ESX1 antisera. Dashes
correspond to molecular mass standards of 110, 68, 45, 31, and 20 kDa.
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The ESX1 derivatives containing the first 68 amino acids (the
N-terminal extension) [ESX1(1-382), ESX1(1-327), and ESX1(1-283)]
generated two polypeptides of approximately equal intensity, which
differed significantly in their mobility following in vitro translation
or transient transfection (Fig.
4B). Because both polypeptides
were
recognized by anti-ESX1 antisera, while only the lower-mobility
ESX1
polypeptides (the upper bands) were recognized by anti-Myc
antisera
(Fig.
4B) (data not shown), we infer that the upper bands
initiate at
amino acid 1 while the higher-mobility polypeptides
(the lower bands)
initiate at the downstream methionine (amino
acid 69). Therefore, the
downstream methionine at amino acid 69,
which is presumed to be the
initiator methionine for transcript
B (Fig.
1A and B), is efficiently
utilized in vitro as well as
in mammalian cells. Inspection of the
amino acid sequence of the
N-terminal extension reveals the presence of
multiple serine and
threonine residues that are putative
phosphorylation sites (Fig.
1A). Furthermore, we have observed that
phosphatase treatment
significantly alters the mobility of the upper
bands (data not
shown). Therefore, the N-terminal extension is likely
to be extensively
modified by phosphorylation, accounting for the
relatively low
mobility of ESX1 proteins containing this
region.
To examine the subcellular localization of the full-length and
truncated ESX1 polypeptides, we transiently transfected the
corresponding expression plasmids into COS-1 cells and examined
their
distribution by indirect immunofluorescence with anti-ESX1
antisera
(Fig.
5). As we observed for endogenous
ESX1 (Fig.
3G
to I), the full-length protein [ESX1(1-382)] was
localized primarily
to the cytoplasm of transfected cells and did not
overlap with
the DAPI nuclear staining (Fig.
5C and D). The N-terminal
extension
or other N-terminal sequences did not influence the
subcellular
localization of ESX1, since polypeptides lacking these
N-terminal
regions [ESX1(69-382) and ESX1(160-382)] were also
primarily cytoplasmic
(Fig.
5E, F, K, and L). In contrast, ESX1
polypeptides lacking
C-terminal sequences, in particular the PF/PN
motif [ESX1(1-327)
and ESX1(1-283)], exhibited nuclear
localization, which overlaps
with the DAPI staining (Fig.
5G to J).
Taken together, these findings
suggest that the PF/PN motif masks or
inhibits the nuclear localization
signal(s) located in other regions of
the ESX1 protein.
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FIG. 5.
The PF/PN domain inhibits nuclear localization of ESX1
in transfected cells. A control plasmid (Vector) or pcDNA3-Esx1
plasmids encoding the indicated proteins were transfected into COS1
cells, and the ESX1 proteins were visualized by indirect
immunofluorescence with anti-ESX1 antisera ( -Esx1) (A, C, E, G, I,
and K), while cell nuclei were visualized by DAPI staining (B, D, F, H,
J, and L). For each pair of images, arrows point to the same cell
showing the anti-ESX1 antiserum staining (green) or the DAPI staining
(blue). Note that ESX1 proteins containing the PF/PN domain
[ESX1(1-382), ESX1(69-382), and ESX1(160-382)] are primarily
cytoplasmic whereas those lacking this domain [ESX1(1-327) and
ESX1(1-283)] are primarily nuclear. Bar, 25 µm.
|
|
The PF/PN motif inhibits the DNA binding activity of ESX1.
To
date, few members of the paired-like class of homeodomains
have been characterized in terms of their DNA binding properties. Moreover, although the ESX1 homeodomain displays the defining sequence
features of the paired-like class, it has only 62% amino acid identity to the most closely related homeodomains (7) and does not contain a conserved region (the paired-tail
motif) that defines a large subclass of paired-like proteins
(32). Furthermore, inspection of the ESX1 sequence reveals
an unusual feature of its homeodomain, namely, a proline at position 43 (Fig. 1A), which is rarely found in homeodomains (7) and
would be expected to truncate the DNA recognition helix by three
residues. Therefore, we performed electrophoretic mobility shift assays to examine the DNA binding properties of the full-length and truncated ESX1 polypeptides produced by in vitro translation (Fig.
6). For this purpose, we used DNA sites
that are known to interact with members of several classes of
homeodomains (sites 6, 6-5, and 6-11) (9) or with members of
the paired (site P2) (53) or paired-like (site PRDQ9) (45) classes of
homeodomains, or a control site lacking the TAAT core that is required
for homeoprotein-DNA binding (site 6-19) (9).
View larger version (72K):
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|
FIG. 6.
The PF/PN domain inhibits DNA binding of ESX1 proteins.
Electrophoretic mobility shift assays were performed as described
previously (9), using increasing amounts (3 or 6 µl;
triangle) of the indicated ESX1 proteins obtained by in vitro
translation. The DNA sites used are indicated; their sequences are
provided in Materials and Methods. The specific protein-DNA complexes
are indicated by asterisks. NA, unprogrammed reticulocyte lysate.
|
|
In general, the DNA binding activity of full-length ESX1
[ESX1(1-382)] was relatively weak and was detectable only with DNA
site 6 (Fig.
6A). Moreover, no DNA binding activity was observed
for an
ESX1 protein lacking the N-terminal extension [ESX1(69-382)]
or one
lacking most of the N-terminal region [ESX1(160-382] (Fig.
6A).
However, an ESX1 protein lacking the PF/PN domain [ESX1(1-327)]
bound avidly to DNA while retaining a preference for binding to
site 6 (Fig.
6). A truncated polypeptide corresponding to the
homeodomain
[ESX1(184-246)] bound to DNA with greater avidity
although with
broader specificity, since it interacted to various
degrees with all of
the DNA sites except the control site lacking
the TAAT core (site
6-19).
Taken together, these data reveal several notable features of the DNA
binding properties of ESX1. First, these findings indicate
that ESX1
interacts specifically with DNA, albeit with low affinity,
and
with a distinct binding specificity from that of other
paired-like homeoproteins, since it binds preferentially to
site 6 rather
than to sites P2 and/or PRDQ9. Second, N-terminal protein
regions
of ESX1 appear to contribute to its overall binding specificity
and affinity for DNA. Third, ESX1 appears to bind to DNA as a
monomer,
in contrast to other
paired-like homeodomains, which
bind as
dimers (
45,
53), as is apparent in Fig.
6 and confirmed
by
more extensive mixing experiments (data not shown). Finally,
and most
notably, our results reveal a second potential function
for the PF/PN
motif, which is to inhibit the DNA binding activity
of
ESX1.
The ESX1 protein contains an SH3 binding motif.
The SH3 domain
is a modular protein interaction domain found in a variety of proteins,
including many involved in signal transduction (reviewed in references
12 and 47). This domain
recognizes short proline-rich sequences that consist of a PXXP core
flanked by other residues, frequently prolines and basic amino acids, which confer binding specificity (18, 27, 36, 47, 54). Inspection of the ESX1 proline-rich region revealed one sequence with
an excellent match to the c-abl SH3 binding consensus (E#1, PPFRPPPLPPF) (36), as well as two other sequences that
contain a PXXP core but whose flanking amino acids do not appear to
correspond to SH3 binding motifs (E#2, PPFPWPPVPPD; E#3, YPMVPRPMHPQ)
(Fig. 1A and B). Notably, the sequence of ESX1 peptide E#1 is similar to that of the SH3 binding domain of 3BP-1 (APTMPPPLPP), which was
isolated on the basis of its interaction with c-abl
(11, 36); however, ESX1 peptide E#1 lacks a leucine at
position 6 that is strongly preferred for binding to the
c-fyn and c-src SH3 domains (37,
46). Moreover, ESX1 peptide #1 has an arginine at position 4, which is frequently found in SH3 binding peptides that bind in the
class I orientation, and it has been suggested to interact with a
corresponding conserved aspartic acid residue in the SH3 domain
(18, 27). While the sequence determinants of SH3 binding
specificity are not yet completely defined, it is clear that nonproline
residues confer specific recognition of distinct SH3 domains
(12). In particular, polyproline stretches such as those
found in many transcription factors do not bind SH3 domains in vitro
(36).
To investigate whether these proline-rich ESX1 peptides bind to SH3
domains in vitro, we performed Far-Western interaction
experiments with
GST fusion proteins containing E#1, E#2, and
E#3 and radiolabeled SH3
domains from c-
abl, c-
fyn, or N-
src
(Fig.
7A and B). We found that ESX1
peptide E#1, but not E#2 or E#3,
binds strongly in vitro to the
c-
abl SH3 domain, weakly to the
c-
fyn SH3 domain,
and not at all to the N-
src SH3 domain (Fig.
7B). We next
asked whether the full-length or truncated ESX1 proteins
exhibit SH3
binding activity by using GST interaction assays performed
with the
GST-c-
abl and GST-c-
fyn fusion proteins and
35S-labeled ESX1 proteins obtained by in vitro translation
(Fig.
7C). We found that each of the ESX1 proteins that contain the
proline-rich region, including peptide E#1 [ESX(1-382), ESX(69-382),
ESX(1-327), and ESX(160-382)], interacted in vitro with the
c-
abl SH3 domain and to a lesser extent with the
c-
fyn SH3 domain (Fig.
7C). In contrast, an ESX1 protein
lacking the proline-rich region
[ESX1(1-283)] did not interact with
these SH3 domains (Fig.
7C).
Thus, the proline-rich region of ESX1
mediates specific interactions
with SH3 domains in vitro.
View larger version (50K):
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|
FIG. 7.
The proline-rich region of ESX1 confers binding to SH3
and WW domains. (A) SDS-PAGE analysis of total-cell lysates containing
GST ESX1 fusion peptides (GST E#1, GST E#2, or GST E#3) and GST SH3
domain fusion proteins (GST c-abl, GST c-fyn, or
GST N-src), visualized by staining with Coomassie blue.
Markers (M) are 66, 45, 36, 24, 20, and 14 kDa. (B) Far-Western
analysis of the interaction of GST ESX1 fusion peptides with
radiolabeled SH3 domains. Each lane contains equal amounts (15 µg) of
a total-cell lysate containing GST ESX1 fusion peptides (GST E#1, GST
E#2, or GST E#3) probed with the indicated radiolabeled GST SH3 domain
fusion proteins. M denotes the lane containing molecular mass markers.
(C) GST interaction assays with radiolabeled in vitro-translated ESX1
proteins (input), followed by incubation with GST alone, with GST fused
to SH3 domains (c-abl or c-fyn), or with GST
fused to WW domains (FBP11WW or FBP30WW). Proteins were visualized by
SDS-PAGE followed by autoradiography. Molecular mass standards (130, 90, 66, 45, 36, and 24 kDa) are indicated by dashes.
|
|
The sequence determinants for binding to the c-
abl SH3
domain are similar to those for binding to several proteins containing
the WW domain, a protein interaction domain named for its
characteristic
two tryptophan residues (
3,
47). Therefore,
we also tested
the ability of ESX1 to interact in vitro with selected
WW domains
by using a GST interaction assay. Since the binding
specificities
of WW domains have not been as well studied as those of
SH3 domains,
we used GST fusions with WW domains from FBP11 and FBP30,
which
have distinct binding specificities (
3). We found that
ESX1
polypeptides containing the proline-rich region
[ESX1(1-382),
ESX1 (69-382), ESX1(1-327), and ESX1(160-382)]
interacted strongly
with the GST-FBP30 WW fusion protein and weakly
with the GST-FBP11
WW protein (Fig.
7C) whereas an ESX1 protein lacking
the proline-rich
region [ESX1(1-283)] did not interact with these WW
domains (Fig.
7C). Taken together, these findings demonstrate that the
proline-rich
region of ESX1 modulates specific interactions with both
SH3 and
WW domains, raising the possibility that ESX1 is involved in
signaling
interactions mediated through such protein-protein
interactions.
| |
DISCUSSION |
Esx1 is one of the few regulatory genes known to have
specific expression and function in the placenta during development. The present study has revealed several novel features of the expression pattern and biochemical functions of the ESX1 protein. First, we have
found that ESX1 is localized primarily to the cytoplasm in the
labyrinth layer of the placenta in vivo. Second, we have shown that
ESX1 contains a novel protein motif, termed the PF/PN domain, which
inhibits both nuclear localization and DNA binding activity. Finally,
we have found that ESX1 contains a proline-rich region that mediates
specific interactions with the c-abl SH3 domain as well as
certain WW domains. These findings suggest that the essential function
of ESX1 in the placenta during embryogenesis may be mediated by its
ability to relay signaling events in the cytoplasm to transcriptional
activity in the nucleus and may be regulated through the inhibitory
action of the PF/PN domain.
The subcellular localization of ESX1 suggests its regulated nuclear
entry.
In contrast to the numerous studies that have examined the
expression of homeobox genes during development, fewer studies have
investigated the expression patterns and/or subcellular localization of
their corresponding protein products. Most of the homeoproteins that
have been examined are localized to the nucleus, consistent with their
roles as putative transcription factors (see, e.g., references
14, 48, and 51). However, some
notable exceptions include the murine Emx1 homeoprotein, which exhibits
nuclear localization early in development but is localized to olfactory
axons in the nervous system at later stages (6). Moreover,
the vertebrate Engrailed homeoproteins are localized to nuclei as well
as caveola-like vesicles, where they are thought to relay signals from
axon terminals to the nucleus (22, 30). In addition, members
of the Extradenticle/PBX class of homeoproteins are found in the
cytoplasm as well as in the nucleus, due to competition between active
nuclear export through a CRM-dependent mechanism and nuclear import by
association with the essential cofactors homothorax and PREP1 (1,
4, 31, 38). In each of these cases, the extranuclear localization of these homeoproteins is presumed to reflect their regulated nuclear
entry and subsequent function as transcription factors. On the other
hand, certain homeoproteins are known to have functions in the
cytoplasm for posttranscriptional control; in particular, Bicoid
functions as a translational regulator through binding to mRNA, in
addition to its activity as a transcription factor (17, 39).
Our observation that the ESX1 protein is primarily cytoplasmic in vivo
most probably reflects regulated nuclear transport
and subsequent
transcriptional activity, since it is also localized
to the nucleus in
a subset of the labyrinth cells of the placenta.
Furthermore, ESX1
contains a nuclear localization signal(s) that
is masked or inhibited
by the PF/PN domain in cell culture. However,
we cannot rule out the
possibility that the cytoplasmic localization
reflects an alternative
function of ESX1, other than (or in addition
to) its presumed activity
as a transcription factor. In this regard,
it is notable that ESX1 is
relatively inefficient at DNA binding
and that it contains an SH3
binding motif, which is often found
in signaling molecules. Although
our data do not distinguish between
retention of ESX1 in the cytoplasm
and its active export from
the nucleus, the inhibitory activity of the
PF/PN domain suggests
a novel mechanism for regulating nuclear
entry.
Potential roles of the unusual protein motifs in ESX1.
ESX1
contains at least two protein motifs that are not generally found in
homeoproteins or other transcription factors. The first unusual feature
is the presence of an SH3 binding motif that can interact in vitro with
the c-abl SH3 domain as well as certain WW domains. While
the precise in vivo target(s) for this interaction remains to be
identified, the ability of ESX1 to bind to the c-abl SH3
domain in vitro indicates its potential to interact with related SH3
domains in vivo. It is noteworthy that a proline-rich domain containing
potential SH3 binding motifs has been noted in another
paired-like homeoprotein, Shox, although it is
unknown whether it binds to SH3 domains (35).
Both the well-studied SH3 domain and the more recently identified WW
domain represent protein interaction modules that participate
in a
broad range of cellular signaling pathways. While SH3 domains
are found
predominantly in cytoplasmic proteins, they are occasionally
encountered in proteins that reside in or transit into the nucleus,
such as c-
abl (
41). Indeed, SH3 domains may be
used to direct
proteins to specific subcellular locations, as in the
case of
the adapter protein GRB-2 and phospholipase C-
(
2). Furthermore,
it has been suggested that certain SH3
domain interactions can
relay cytoplasmic signaling events to the
nucleus, as is the case
for the interaction between c-
vav
and heterogeneous ribonucleoprotein
K (
21).
A second unusual feature of ESX1 is the PF/PN motif, which we have not
found in other proteins in sequence databases. This
peculiar motif
consists of an alternating repeat of proline and
phenylalanine residues
followed by proline and asparagine residues
(Fig.
1A), which is
predicted to assume a flexible coiled-coil
secondary structure. The
PF/PN motif may provide an effective
means of regulating the
transcriptional activity of ESX1, since
it can inhibit both nuclear
localization and DNA binding activity.
We propose that the inhibitory
role of the PF/PN motif may be
mediated by intramolecular interactions
with the homeodomain and
other protein regions responsible for nuclear
localization and
DNA binding and/or by interference with SH3 or WW
domain binding
by the proline-rich region. In this regard, we speculate
that
binding to an SH3 or WW domain-containing protein may unmask
nuclear
localization signals, allowing transport of ESX1 to the nucleus
and subsequent transcriptional
activity.
In addition to its unusual protein motifs and atypical homeobox
sequence, other novel properties of
Esx1 suggest its recent
evolutionary origin.
Esx1 is an X chromosome imprinted gene
(
25),
which is notable since genomic imprinting arose during
mammalian
evolution and is intimately associated with placental
development
(
49). The recent evolution of chorioallantoic
placentation may
have allowed the utilization of novel molecular
mechanisms and
signaling pathways that have not been encountered in the
better-conserved
processes that underlie fetal
development.
| |
ACKNOWLEDGMENTS |
We thank Philip Leder, in whose laboratory this work was
initiated, for his advice and generous support. We also thank Mark Bedford and Philip Leder for the WW domain fusion proteins. We acknowledge the contributions of Lu Yang, Hongyu Wang, Peter
Sciavolino, and David Chan during the initial stages of this work. We
are also grateful to Thomas Bürglin for advice on library
screening, Chaosu E for assistance with DNA sequencing, and Drew
Vershon for helpful comments on the manuscript.
S.M.S. was supported by a predoctoral fellowship (96-2003-CCR-00) from
the New Jersey Commission on Cancer Research. C.A.-S. was supported by
NIH grant HD33362, and M.M.S. was supported by a Leukemia Society of
America Special Fellowship and by NIH grant HL60212.
Y.-T.Y., S.M.S., and J.D. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for M.M.S.: CABM,
679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5645. Fax: (732) 235-5318. E-mail: mshen{at}cabm.rutgers.edu. Mailing
address for C.A.-S.: CABM, 679 Hoes Ln., Piscataway,
NJ 08854. Phone: (732) 235-5161. Fax: (732) 235-4850. E-mail:
abate{at}cabm.rutgers.edu.
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Molecular and Cellular Biology, January 2000, p. 661-671, Vol. 20, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
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