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From the
Received for publication, September 6, 2001, and in revised form, December 10, 2001
The transcription factor AP-2 The family of AP-2 transcription factors consists of four
different genes known as AP-2 During development, expression of AP-2 In the study presented here we used a combination of
suppression-subtractive hybridization (12) and high throughput
differential screening to identify target genes of transcription factor
AP-2 The genes are KLF-4
(Krüppel-like factor 4 (13,
14), mEFEMP-1 (epidermal growth
factor-containing fibulin-like extracellular matrixprotein 1, (15)), Mtd
(Matador (16)), and
Stra13 (Stimulated by retinoic
acid 13 (17)). RT-PCR analyses confirmed that the genes
were derepressed in the mutants. Most notably KLF-4 could be detected
as early as E 8.5 in the mutant mesenchyme, whereas in wild-type
animals KLF-4 can first be detected at E 10.5. Expression of the
KLF-4 gene had been shown to induce cell cycle exit and terminal differentiation (18). In fact, fibroblasts derived from
AP-2 Isolation of RNA--
Poly(A)+ RNA was prepared from
a single embryo head at E 8.75 (exactly 18 somites) from a wild-type
and an AP-2 Synthesis of cDNA--
First and second strand synthesis was
done using the SMART cDNA synthesis kit
(CLONTECH, USA) after thorough optimization of the
cycle number. To test the PCR being in the logarithmic phase, 1/10 of
the cDNA was labeled and electrophoresed on an alkaline agarose
gel. 50% of the poly(A)+ RNA was subjected to 25 cycles of
PCR according to the manufacturer's instructions.
Subtractive Hybridization--
Isolation of AP-2
For the first hybridization the mixture of driver and tester, cDNA
was denatured at 100 °C for 20 s and then cooled over 1 min to
68 °C, and temperature was maintained for 8 h. For the second
hybridization, driver cDNA was denatured at 100 °C for 20 s
and then added to the pooled mixture of the previous hybridization and
incubated at 68 °C for 20 h. Modifications of the
manufacturer's protocol were done according to published procedures
(19).
Subtraction Efficiency--
The PCR-amplified cDNA was
digested with RsaI (a 4-base cutter) and HindIII
linkers (HindIII linker, 5'-ATCGTCAAGCTTCAAGTTAGCATCG-3' and
5'-GCTAACTTGAAGCTTGACGAT-3') were ligated to the tester cDNA, and
EcoRI linkers were ligated to the driver cDNA
(EcoRI linker, 5'-TAGTCCGAATTCAAGCAAGAGCACA-3' and
5'-CTCTTGCTTGAATTCGGACTA-3'). Free linkers were removed, and 1/500 of
the ligated cDNA mixture was amplified with the appropriate primers
in standard buffer (Amersham Biosciences AB) with 2 units of
Taq polymerase (30 cycles at 94 °C, 20 s; 52 °C,
20 s; and 72 °C, 2 min). For the subtracted cDNA, a nested
PCR approach was used with the following conditions: 1st PCR, 27 cycles
at 94 °C, 20 s; 68 °C, 20 s; 72 °C for 2 min; 2nd
PCR, 12 cycles at 94 °C, 20 s; 68 °C, 20 s; 72 °C
for 2 min. Equal amounts of the amplified material was run on an
agarose gel (visualized under a UV transilluminator after staining with ethidium bromide), blotted, and subjected to hybridization under stringent conditions.
Differential Screening via Reverse Northern--
A total of 4800 individual clones was picked and used to inoculate 96-well microtiter
plates containing LB media supplemented with 100 µg/ml ampicillin.
After incubation on a gyratory shaker for 4 h, a 10-µl aliquot
was transferred to 50 µl of distilled water in PCR tubes. The
bacteria were lysed by heating to 100 °C for 5 min, and then 40 µl
of distilled water was added. Samples of 10 µl were used to amplify
the cDNA inserts using M13 standard primers under standard
conditions. (Note: clones containing no insert also did produce a band
on the gel equal to the multiple cloning site.) The PCR products were
run in duplicate on high density agarose gels (Centipede; Owl
Scientific, Woburn, MA). PCR-amplified AP-2
All hybridizations were carried out in Church hybridization buffer at
65 °C. The filters were exposed to x-ray film, and the respective
bands were compared and referenced to loading position and intensity of
the controls (AP-2 Virtual Northern--
Virtual Northern blots were performed
according to the manufacturer's instructions (SMART and PCR-Select
subtraction manuals, CLONTECH). 500 ng of
unsubtracted SMART cDNA and 500 ng of SMART cDNA after
PCR-Select subtraction derived from both wild type and
AP-2 Isolation of Full-length cDNA--
For isolation of
full-length cDNAs, two mouse embryo libraries (E 8.5 and E 10.5, Invitrogen) were pooled and plated. Filter lift assays were performed
with pools of four recombinant fragments each. Colonies, which gave a
positive signal, were picked using 1 ml of sterile Eppendorf tips,
diluted in LB, and streaked onto 10-cm bacterial dishes. Filter lifts
of these were cut into quarters and probed with individual fragments.
Positive clones were picked, and the insert size was evaluated by
restriction digest.
Whole Mount in Situ Hybridization--
Digoxigenin-labeled
probes were generated using recombinant clones containing full-length
cDNAs with T7 and SP6 RNA polymerase. E 8.5-10.5 embryos were
hybridized according to standard protocols as previously described (20)
(stratus.lifesci.ucla.edu/hhmi/derob).
Preparation of Murine Fibroblasts and Analysis--
Murine
fibroblasts were derived from timed matings at embryonic days
13.5-15.5. The head was cut off, and the internal organs were removed
and used for genotyping. The embryo carcass was minced using fine
scissors and digested with trypsin. After centrifugation and removal of
cellular debris, cells were seeded on a 15-cm tissue culture dish
(Greiner, Germany) and cultured in Dulbecco's modified Eagle's medium
(Invitrogen) with 10% fetal calf serum (Biochrom, Germany). The cells
were passaged 3 times to obtain a pure population of fibroblast cells.
For growth curve analysis, 104 cells of each genotype were
plated in a 6-cm tissue culture dish with gridlines (NUNC cell culture
dish with a 2-mm grid, Nalgene-NUNC), and four individual grids were
counted at the time indicated. For Northern analysis a 10-cm tissue
culture dish of fibroblasts of passage 2 was harvested, and 10 µg of
total RNA was loaded on an agarose gel, electrophoresed, and blotted on
a nylon membrane. Hybridization was performed according to standard
procedures with [32P]dCTP-labeled PCR fragments of probes indicated.
PCNA Staining--
Fibroblasts of passage 2 were plated on
chamber slides (Lab-Tek II chamber slides, Nunc) and allowed to adhere
overnight. After rinsing the cells once with PBS they were fixed with
10% formalin. After rinsing twice with distilled H2O, the
cells were incubated in 2 N HCl for 10 min and then washed
with distilled H2O and incubated in methanol, 0.3%
H2O2 for 20 min. After rinsing in distilled
H2O and PBS, two times respectively, the cells were blocked
in PBS plus 10% horse serum plus 0.5% mouse serum for 10 min. Then
they were rinsed in PBS and incubated with PCNA antibody diluted to
1:100 in PBS, 0.1% Triton X-100 (Dako, Hamburg, Germany) for 1 h.
After rinsing twice with PBS the cells were incubated with secondary
antibody (1:50 in PBS) developed using the manufacturer's conditions
(Vector ABC-Elite and AEC Kits) and counterstained with hematoxylin for
30 s. Slides were embedded in immumount (Shandon-Lipshah, UK) and
photographed using a Prog.Res. digital camera (Jenoptik, Germany) fitted to a Zeiss-Axiovert microscope (Zeiss, Germany).
Generation of cDNA Libraries of a Single Head of Wild-type and
AP-2
After the forward- and reverse-subtracted cDNA libraries were
generated, they were tested for the efficiency of the subtraction. The
degree of subtraction can be determined by monitoring the enrichment of
sequences specific to one population after subtraction and depletion of
transcripts common to both populations. The G418 resistance gene,
introduced to inactivate the AP-2 Screening of 4800 Colonies Yields 52 Differentially Expressed
Genes--
For the identification of genes regulated by AP-2 Many of the Clones Represent Novel Sequences--
All 52 clones
were sequenced and analyzed using the BLAST server to determine the
nature of the sequence. As depicted in Table I, sequence analysis showed that the 52 clones encode for 46 different genes (25 known and 21 novel cDNAs)
indicating that we had isolated some genes several times.
Isolation of Full-length cDNA Clones--
Generation of
cDNA pools with SMART (CLONTECH) results in
average fragment lengths of 300-600 base pairs. To clone full-length cDNA of the clones, we screened a commercial cDNA library stage E 8.5 and E 10.5 (Invitrogen). 500,000 clones were hybridized with
pools of four fragments. Positive signals were picked and rescreened
with the individual fragment. After determination of the insert size
the clones were used for sequence analysis.
The full-length clones obtained were subjected to a whole mount
in situ screen to detect genes expressed in structures
affected by the AP-2
The transcriptional repressor Stra13 had been described to
be expressed at day E 9.5 in Rhombomere 5 (17). We were able to
reproduce these data and moreover detected Stra13 RNA in tip of the
tail and in the mesenchyme surrounding the dorsal aorta at E 9.5 (Fig.
1E, arrowheads). Stra13 had been reported to
induce growth arrest and terminal differentiation in cell culture
(17).
Our screen resulted in cloning of the murine homologue of
EFEMP-1, an extracellular matrix protein that was found
overexpressed in senescent and quiescent fibroblast cultures and a
patient with Werner syndrome, a genetic disorder characterized by
accelerated aging (15). We see mEFEMP-1 in cells of the
paraxial mesenchyme in the hindbrain and in a fine, distinct line in
the trunk (Fig. 1F, arrow).
Mtd (or bok1), a member of the Bcl-2 family, had
been described to be expressed in the brain, liver,
and lymphoid tissues and to activate apoptosis (16, 21, 22). Our
in situ analysis revealed expression in limb bud and
paraxial mesenchyme in the trunk and craniofacial mesenchyme at E 10.5 (Fig. 1G, arrowheads).
Krüppel-like factor-4, KLF-4, is published to be
expressed in the epithelial cells of the mucosa of the gut (23).
However, we see expression in a highly regionalized pattern in the
frontonasal area, the first and second branchial arch, the apical
ectodermal ridge of the limb buds, and the dorsal root ganglia in the
trunk (Fig. 1H, arrowheads). Many lines of
evidence suggest that KLF-4 expression is indicative for growth arrest
and differentiation. For example, NIH 3T3 cells are found to express
KLF-4 under serum deprivation or upon contact inhibition (18). PMT-3
cells, which are transfected with a plasmid expressing KLF-4, show
reduced thymidine uptake as well as greatly diminished DNA synthesis as determined with bromodeoxyuridine labeling (14).
Taken together, the genes repressed by AP-2 Four Target Genes Are Expressed Prematurely in the Mutant
Animals--
We had subjected a subtracted library from E 8.75 wild
type and mutant material to an in situ screen, which was
aimed to detect genes that display a distinct and overlapping
expression pattern compared with AP-2 In Mutants Premature Expression of KLF-4 Is Localized to the
Mesenchyme--
To be considered as a direct target of a transcription
factor, the gene in question has to be expressed in the same cells. At
E 8.5 AP-2 Embryonic Fibroblasts Express KLF-4--
We next established
primary fibroblast cultures from E 13.5 to 15.5 embryos to substantiate
the findings from the RT-PCR and in situ analyses. RNA was
prepared from control and mutant cultures that had been passaged three
times to obtain a pure population of fibroblast cells. We could not
detect signals for Mtd or mEFEMP-1 (not shown),
and the signal of Stra13 was comparable from wild type to
mutant animal (Fig. 2A), but KLF-4 was found to
be induced in mutant fibroblast cells (Fig. 2A). This result
demonstrates that the derepression could also be found in the in
vitro system tested.
AP-2-deficient Embryonic Fibroblasts Are Growth-retarded--
We
decided to utilize this cell system to exploit further the functional
consequences of this altered gene expression. Transfection of
KLF-4 cDNA in NIH 3T3 fibroblast culture had been described to result in reduced proliferation (18). Based on these results, we
reasoned that fibroblast cultures from AP-2
This difference in growth may be due to enhanced apoptosis or reduced
proliferation. We performed annexin V staining of wild type and mutant
cultures and found comparable levels of apoptosis in both cultures (not
shown). This result is in agreement with our finding that
Mtd, the target gene inducing apoptosis, is not found
expressed in fibroblast cells of either genotype.
To test for proliferating cells, we stained wild type and mutant
cultures with the PCNA antibody. We found that only a fraction of the
mutant cells were PCNA-positive, whereas in the wild type controls
nearly all of the cells were positive for this marker (Fig. 3,
C and D). This result shows that the growth
retardation seen in mutant cell lines originates from reduced
proliferation but not enhanced apoptosis.
Analysis of Promoter Sequences Reveals Potential Binding Sites for
AP-2 Expression of AP-2 We have been isolating target genes of transcription factor
AP-2 The cranial closure defect in AP-2 The role for proliferation is further supported by the fact that many
human mammary tumors and cell lines derived from tumors display
overexpression of AP-2 genes (28). Furthermore, AP-2 is able to activate c-erb-B2, a receptor tyrosine kinase
implicated in cellular proliferation (1). In fact, AP-2 With the results of the screen for target genes of AP-2 Data from Drosophila AP-2 (dAP-2 (34, 35)) support the
function of AP-2 as being a regulator of cell growth. DAP-2
is expressed in the cells of the presumptive leg joints, and flies mutant for dAP-2 show a severe reduction in leg length. They fail to
produce joint structures, indicating that dAP-2 expressed in joint
cells is able to influence growth of leg segments (36, 37).
Overexpression of dAP-2 does not induce overgrowth of the limb. In the
model put forward by Kerber et al. (36) dAP-2
would be needed to promote cell growth and cell survival. They
speculate that dAP-2 might activate a cellular survival factor. Thus
dAP-2 and AP-2 The question remains whether KLF-4, Mtd,
Stra13, and mEFEMP-1 are directly regulated by
AP-2 The role of the requirement of KLF-4 deregulation in AP-2 Taken together, the family of AP-2 transcription factors might play a
role in the well orchestrated phases of proliferation of various tissue
types where they function as control factors that inhibit the
expression of differentiation associated genes and enable the
expression of growth-promoting genes.
We thank Tanja Schüler, Yvonne
Petersen, Richard Jäger, and Oliver von Stein.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by Deutsche Forschungsgemeinschaft Grant 503/2. To
whom correspondence should be addressed: Dept. of Developmental Pathology, Institute of Pathology, Bonn University, Sigmund-Freud Strasse 25, 53127 Bonn, Germany. Tel.: 49-228-287-6342; Fax:
49-228-287-5030; E-mail: Hubert.Schorle@ukb.uni-bonn.de.
Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M108578200
The abbreviations used are:
E, embryonic
development;
GAPDH, glyceraldehyde phosphate dehydrogenase;
PBS, phosphate-buffered saline;
PCNA, proliferating cell nuclear
antigen;
RT, reverse transcriptase.
A Subtractive Gene Expression Screen Suggests a Role of
Transcription Factor AP-2
in Control of Proliferation and
Differentiation*
,, and§¶
Forschungszentrum Karlsruhe, ITG, Hermann
von Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen and the
§ Department of Developmental Pathology, Institute of
Pathology, Bonn University, Sigmund-Freud Strasse 25,
53127 Bonn, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has
been implicated as a cell type-specific regulator of gene expression
during vertebrate embryogenesis based on its expression pattern in
neural crest cells, ectoderm, and the nervous system in mouse and frog
embryos. AP-2 is prominently expressed in cranial neural crest
cells, a population of cells that migrate from the lateral margins of the brain plate during closure of the neural tube at day 8-9 of embryonic development. Homozygous AP-2 mutant mice die
perinatally with cranio-abdominoschisis, full facial clefting, and
defects in cranial ganglia and sensory organs, indicating the
importance of this gene for proper development. By using a subtractive
cloning approach, we identified a set of genes repressed by AP-2
that are described to retard cellular proliferation and induce
differentiation and apoptosis. We show that these target genes are
prematurely expressed in AP-2 mutant mice. One of the genes
isolated, the Krüppel-box transcription factor KLF-4
implicated in induction of terminal differentiation and growth
regulation, is found expressed in mutant embryonic fibroblasts. We show
that fibroblasts lacking AP-2 display retarded growth but no
enhanced apoptosis. Based on these data we suggest that
AP-2 might be required for cell proliferation by
suppression of genes inducing terminal differentiation, apoptosis, and
growth retardation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, -
, and
-
, respectively (Tcfap2a, Tcfap2b, and
Tcfap2c-Mouse Genome Informatics) (1-7). AP-2 transcription factors homodimerize using a unique C-terminal helix-span-helix motif.
Sequence-specific binding to the consensus GCCN(3/4)GGC is mediated by
a basic domain overlapping with the dimerization domain. The
transcriptional activation is mediated by a proline/glutamine-rich cassette at the N terminus (6). These domains are highly conserved in
all three known AP-2 genes.
initiates around
day 8 of embryonic development
(E1 8.0) in premigratory
neural crest cells (8). During organogenesis expression of
AP-2 can be detected mainly in neural crest-derived tissue, kidney, and skin. Gene knockout experiments have shown that
disruption of the AP-2 gene leads to a very severe body wall and neural tube closure phenotype with homozygous animals dying
perinatally (9, 10). The mutants show a hypoplasia of the cranial
ganglia and high degree of apoptotic cranial neural crest cells
indicating the importance of AP-2 for craniofacial development (9).
Although a variety of genes have been proposed as targets for
transcriptional regulation of AP-2 (reviewed in Ref. 11) based on
studies in cell culture systems, the pathways regulated by AP-2 in
the process of craniofacial morphogenesis are poorly understood.
. By using a single head of an E8.75 knockout and a control
animal, we generated cDNA, which was subjected to
suppression-subtractive hybridization. The success of the subtraction
was demonstrated by virtual Northern blots with different known genes.
We subjected 4800 recombinant clones to two rounds of differential
screening using reverse Northern blots, and we isolated a total of 52 recombinant clones. Four genes from the screen for repressed genes,
which we analyzed further, are known to be involved in growth control, differentiation, and apoptotic processes.
mutant animals were shown to express KLF-4
and displayed reduced proliferation. We conclude that
AP-2 might repress a set of target genes, which suppress
cellular proliferation and induce terminal differentiation and
apoptosis. The work outlined in this paper helps in understanding the
molecular processes controlled by transcription factor
AP-2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-deficient specimen according to the manufacturer's
instructions (Invitrogen FastTrack 2.0) with the exception that the
material was homogenized using a 16-gauge syringe before adding the
lysis buffer. The quality of the isolated RNA was determined via RT-PCR
with GAPDH and AP-2 (sequences of the primers available upon request).
up-regulated
target genes was achieved by performing suppressive-subtractive
hybridization protocol between wild type ("tester") and
AP-2-deficient ("driver") cDNA using the PCR-select cDNA
subtraction kit (CLONTECH) with modifications as
described (19) (referred to as forward screen). For genes repressed by
AP-2, the same procedure was used except that cDNA from
AP-2-deficient embryonic heads served as tester, and the wild
type material was used as driver (referred to as reverse screen).
and GAPDH cDNA was
loaded and served as hybridization control. Gels were electrophoresed
for 45 min at 100 V and photographed. The DNA was then denatured for 15 min in 0.4 N NaOH, blotted onto nylon membrane (Hybond N+,
Amersham Biosciences), and hybridized with 32P-labeled
cDNA probes from wild type and mutant, respectively. For the
labeling reaction 1/10 of the poly(A)+ RNA (Invitrogen
FastTrack 2.0) from wild type and mutant embryonic heads stage E 8.75 was subjected to 15 cycles of PCR in the presence of
[32P]CTP according to the manufacturer's instructions
(SMART, CLONTECH).
as well as GAPDH was run twice on each gel).
-deficient mRNA was separated on an agarose gel, stained with EtBr, and transferred onto a nylon membrane (Hybond N, Amersham Biosciences). The blots were then hybridized to
[32P]dCTP-labeled PCR fragments of GAPDH and neomycin
resistance gene under Church buffer conditions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Mutant Embryos at E 8.75--
AP-2 mutant animals fail to
close the cranial neural tube, an event that takes place between E 8.5 and 9.5 of murine development. The expression of AP-2 is first
detected at E 8.0 in the apical neural fold and the lateral head
mesenchyme. To isolate genes involved in the initial steps of the
developing phenotype, we isolated embryos at age E 8.75. To ensure that
we were using same age material, the somites were counted. Each embryo
was genotyped using yolk sac DNA and PCR primers specific for the
AP-2 locus. By using 18-somite embryos, RNA was generated
from a single head of a wild-type as well as a knockout specimen. Then
cDNA was generated and amplified (SMART cDNA synthesis kit,
CLONTECH). These cDNAs were then used for a
PCR-based subtraction, the suppressive-subtractive hybridization
protocol (12) (PCR-Select, CLONTECH). The screen for genes induced by AP-2 is referred to as "forward" screen (RNA from wild type was used as driver and RNA from AP-2-deficient as tester). Genes repressed by AP-2 were identified using RNA from
AP-2 mutants as driver and RNA from wild type as tester, termed
"reverse" screen.
locus, is under ubiquitous
expression driven by the phosphoglyceraldehyde transferase promoter and
thereby serves as an excellent internal positive control that should be
found enriched in the cDNA of the forward screen. Conversely
glyceraldehyde phosphate dehydrogenase (GAPDH, a housekeeping gene)
should be depleted from the subtracted material. GAPDH was shown to be
reduced ~1000-fold in the subtracted compared with the unsubtracted
material of both the forward and the reverse subtractions (Fig.
1A), whereas the neomycin
cDNA was found enriched ~50-fold in the forward direction (Fig.
1B). Taken together, the results indicate that the
population of cDNA had been successfully subtracted, and the highly
expressed housekeeping genes like GAPDH had been eliminated or greatly
reduced. So we could conclude that both subtractions of the cDNA
worked to a high efficiency, and the subtracted cDNA libraries
could be transformed into a cloning vector for the screening
procedure.
View larger version (74K):
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Fig. 1.
Subtraction, high throughput analysis,
and whole mount ISH screen. Equal amounts of PCR-amplified driver
and tester cDNA were run on a 1% agarose gel, blotted, and
hybridized with [32P]dCTP-labeled GAPDH (A)
and G418 resistance cassette (B) probes. Signals of
subtracted and not subtracted material of mutants (ko) and
control (Wt) are shown. C, differential screening
of the subtractive cDNA libraries. Gels were run in duplicate, and
equal loading was checked by ethidium bromide staining using an UV
transilluminator. Filters were then hybridized with
RsaI-digested and radiolabeled cDNA of driver
(D, upper panel) and tester (D,
lower panel). Signals were compared and differences in band
intensity indicated differentially expressed genes (arrows
in D). E-H, whole mount in situ
hybridizations of control embryos stage E 9.5 (E and
F) and 10.5 (G and H). The probes used
were Stra13 (E), mEFEMP-1 (the murine homologue of EFEMP-1;
F), Mtd (G), and KLF-4 (H).
2400 colonies of each subtracted cDNA library were picked and the
cDNA fragments amplified by colony PCR. Each cDNA fragment was
then separated on two high density gels in parallel (Fig.
1C), blotted, and hybridized with radiolabeled cDNA
derived from wild-type (Fig. 1D, upper panel) and
AP-2-deficient embryos (Fig. 1D, lower panel). In the first round we identified 254 clones in the forward and 234 clones in the reverse screen as being differentially expressed. We
re-screened these clones in a second round and used only the clones
with the most striking differences in signal. Taken together, we
performed two rounds of reverse Northern to screen 4800 clones from
both the forward and reverse direction, and we found 52 clones being
differentially expressed. Due to extreme limitation of mRNA material, we performed virtual Northern blots to verify that a given
clone is in fact differentially expressed. Thirty clones were tested
this way (data not shown), and 95% showed the expected differential
expression pattern.
Result of the screen for genes regulated by AP-2
knockout phenotype. Fig. 1 (E-H)
shows a panel of genes that are derived from the screen for repressed
genes, Stra13 (Fig. 1E), mEFEMP-1
(epidermal growth factor-containing fibulin-like extracellular matrix
protein-1, Fig. 1F), Mtd (Matador 1, Fig. 1G), and KLF-4 (Krüppel-like factor 4, Fig.
1G).
retard the cell cycle
and induce terminal differentiation and apoptosis.
. For a target gene to be
involved in the cranio-abdominoschisis, it had not only to be expressed
in the similar structures but also at the gestational time the mutant
embryo would start to display phenotypic alterations. Hence, we decided
to concentrate the next analysis around stages E 8.5-10.5. We
performed RT-PCR analyses of pools of three embryonic heads stage E
8.5-10.5 of wild type and mutants, respectively (Fig.
2A). Three of the four genes
(KLF-4, Mtd, mEFEMP-1, Fig.
2A) tested were found to be expressed prematurely in the
mutant animals; one gene (Stra13, Fig. 2A) seemed
to be expressed more strongly in the mutants. This result demonstrates
that loss of AP-2 leads to a derepression of the target genes in the
mutant embryo.
View larger version (58K):
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Fig. 2.
RT-PCR of KLF-4, Mtd, Stra13, and
mEFEMP-1. A, RT-PCR analyses with primers specific for
KLF-4, Mtd, mEFEMP-1,
Stra13, GAPDH, and tubulin. GAPDH and
tubulin was used to standardize the cDNA used. cDNA was
generated from stages E 8.5 to 10.5 as indicated. The lanes where no RT
material was added ( 
RT) show no signals. Co,
control, water only. B, whole mount in situ
analysis using a digoxigenin-labeled cDNA of KLF-4 on wild-type
(wt) and AP-2-deficient (ko) embryos stage E
8.5. (fb, mb, and hb) forebrain,
midbrain, and hindbrain, respectively.
m-RNA is found in mesenchymal parts of the embryo, mainly
in the cranial mesenchyme of the head folds (8, 9). Because the RT-PCR
analysis had already shown the temporal differences, we performed a
series of whole mount in situ hybridizations to determine
the spatial expression of KLF-4 in the mutants compared with the
wild-type controls. We found KLF-4 expressed in mesenchymal structures
of mutants at stage E 8.5 (Fig. 2B, right),
whereas no signal could be detected in wild type (Fig. 2B,
left). These results indicate that loss of
AP-2 leads to misexpression of KLF-4 as early
as E 8.5.
-deficient animals expressing KLF-4 might display different growth rates compared with
wild-type control cultures. To test this hypothesis, we performed a
cell growth assay. 2 × 104 cells were seeded in a
tissue culture dish and counted after 24, 48, and 72 h according
to standard procedures (24). As seen in Fig.
3B, cell numbers increased
steadily in control cultures (Fig. 3B, wt), in
contrast cells lacking AP-2 were clearly growth-retarded (Fig.
3B, ko).
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Fig. 3.
AP-2 -deficient
fibroblasts express KLF-4. A, Northern blot analysis of
wild type (+/+), heterozygous (+/), and AP-2-deficient (/)
primary murine embryonic fibroblasts. Cells were grown in cell culture
using standard procedures, harvested, and whole RNA prepared. RNA was
probed with KLF-4, MTD, Stra13, and GAPDH as indicated. B,
in vitro proliferation assay. Embryonic fibroblasts were
derived from E 13.5 embryos of all three genotypes. Identical numbers
of cells were plated. The growth of the cultures was measured by
counting the total number of cells on each of the days indicated. Each
line represents a cell line derived from an individual embryo. A set of
representative experiments is shown. Similar results were obtained with
fewer cells plated or higher passage number used (not shown).
Wt, control cells; ko, AP-2-deficient cells.
C and D, cells from experiment B were
subjected to PCNA analysis. Cells undergoing division display the PCNA
antigen and are detectable as red dots. C, PCNA
staining with control cultures; D, PCNA staining with
AP-2-deficient cells.
--
To determine the presence of possible AP-2-binding sites
in the promoter regions of KLF-4, Mtd,
Stra13, and mEFEMP-1 we performed an in
silico search for GCCN(3-4)GGC, the bona fide
AP-2-binding site on genomic fragments spanning the promoters of the
respective genes using the MatInspector version 2.2 program (25).
Analysis of the promoter of KLF-4 (GenBankTM
accession number AF117109) showed that there are at least three
half-sites that fulfill the criteria for AP-2 binding. The promoter
of the human Stra13 gene (GenBankTM accession
number AC090955) shows four AP-2 consensus sequences in close proximity
to the transcriptional start site. A 1.5-kb fragment upstream of the
first exon of Mtd shows at least four AP-2-binding sites
(GenBankTM accession number AC027147). The promoter of
EFEMP-1 (GenBankTM accession number AC096549)
encodes for one AP-2-binding sequence 750 base pairs 5' to exon 1. Taken together, all promoter regions analyzed contain AP-2-binding
elements. Hence a direct regulation of these genes by AP-2 is possible.
Promotes Proliferation and Cell Survival via
Repression of Genes Involved in Terminal Differentiation and
Apoptosis--
Based on the data presented, we would like to present a
model whereby transcription factor AP-2 acts as a suppressor of
KLF-4 (and other target genes such as Mtd,
Stra13, and mEFEMP-1). Because the genes
suppressed are described to induce cell cycle exit and terminal
differentiation, AP-2 might serve as a gatekeeper in controlling the
proliferation versus differentiation of cranial neural crest
cells (Fig. 4). As long as AP-2 is
expressed during the migratory phase of neural crest migration, the
cells are able to proliferate. As soon as the cells reach their
respective targets, AP-2 expression ceases and genes inducing
differentiation would be derepressed (Fig. 4A). Lack of
AP-2 leads to premature expression of AP-2 target genes, which
induce cell cycle arrest, terminal differentiation, and apoptosis (Fig.
4B).
View larger version (42K):
[in a new window]
Fig. 4.
AP-2 acts as
transcriptional repressor to enable proliferation over
differentiation. The effect of expression of transcription factor
AP-2 is shown. The x axis displays the (relative) time a
given neural crest cell will migrate and proliferate (green
bar) or differentiate (red bar). The y axis
displays the (relative) expression of AP-2 (green
line) and the target genes (mEFEMP-1, KLF-4,
Mtd, and Stra13; red line).
A, the expression of AP-2 during, for example, migration
of cranial neural crest cells suppresses the target genes. As
consequence the cells migrate and proliferate. Lack of AP-2
(B) leads to a derepression of the target genes indicated
which leads to a premature differentiation and apoptosis of the
respective cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
involved in craniofacial development. We utilized a
combination of elements of suppression-subtractive hybridization and
high throughput differential screening which permitted the rapid
cloning of rarely transcribed differential genes. We started with a
single head of a wild-type and AP-2-deficient embryo (stage 18 somites) and constructed cDNA libraries, which were subjected to
suppression-subtractive hybridization (12, 26). Control hybridization
showed that the subtraction was successful in that the housekeeping
gene GAPDH was reduced ~1000-fold. This method has several
important features allowing for the successful isolation of AP-2
target genes, which are involved in craniofacial development.
Transcripts that differ more than 5-fold in their abundancy between the
samples to be compared have a higher likelihood to be isolated compared
with transcripts that show only moderate differences in expression (12). The representation of different mRNA species in the
respective pools is being normalized, which increases the relative
representation of rare transcripts (27). This was most likely the
important step because master regulatory genes, such as transcription
factors, are expressed at very low levels compared with housekeeping
genes. We subjected 4800 recombinant clones to two rounds of
differential screening using reverse Northern blots and isolated a
total of 52 recombinant clones. After the screening procedure we came
up with 52 differentially expressed clones, which were then analyzed for their specific expression pattern in whole mount in situ
hybridizations. We show that the technique allows for inexpensive large
scale screen of gene expression programs affected by loss of
AP-2, beginning from minute amounts of tissue. Four genes
from the screen for repressed genes, which we analyzed further, are
known to be involved in cell cycle control, differentiation, and
apoptotic processes. They are discussed below.
mutants becomes evident first
around closure of the neural tube at E 8.5-9.5 (9, 10). Therefore we
decided to look at the expression of these potential target genes of
AP-2 in this time window. We performed RT-PCR of these genes at E
8.5-10.5 from poly(A)+ RNA of wt and mutant embryos. The
experiment could clearly show that the expression of these genes is
altered in the mutant embryos. Stra13, expressed at E 8.5 in control
embryos, was found to be more strongly expressed from E 8.5 on in
the mutants. MEFEMP-1, Mtd, and KLF-4
show a derepression leading to premature expression starting from day E
8.5 in mutants. The transcription factor KLF-4 shows the
most tremendous effect. In control embryos its expression is first
detectable at E 10.5, but in embryos we observe expression already at E
8.5. This finding was further substantiated in whole mount in
situ hybridizations of wt and ko embryos at E 8.5. We show that
lack of AP-2 leads to up-regulation of KLF-4 in cranial mesenchyme and in fibroblast cultures. As a consequence the
proliferation of the fibroblast cells is reduced as determined by PCNA
staining and cell growth assay. Thus, AP-2 might serve as a
gatekeeper in promoting proliferation by suppression of
differentiation in neural crest cells during migration.
had been
discussed as being an oncogene, and transgenic experiments addressing
this issue are underway. Furthermore, AP-2 is expressed in the
mitotically active basal cell layers of the skin but not the terminally
differentiating, nondividing suprabasal layers (29). It is tempting to
speculate that it is in fact repression exerted by AP-2 transcription
factors that keeps the cells of the basal layer in the cell cycle and prevents premature differentiation. Although AP-2-deficient mice do
not display defects in the cells of the skin, loss of its function might be compensated by AP-2, which is expressed strongly in basal
cells (30).
, the failure
of the cranial neural tube to close in mice lacking AP-2 might be
explained. Based on a model put forward by Schoenwolf and co-workers,
neural tube, surface ectoderm, and cranial mesenchyme act in an
orchestrated way to bring about the closure of the neural tube
(31-33). Failure of one component results in failure of the neural
tube to close. We speculate that due to the lack of AP-2, cells of the cranial mesenchyme express differentiation and growth retarding genes prematurely. As a consequence the proliferation at the
time of cranial neural tube closure (E 8.75) is reduced resulting in
hypoplastic mesenchyme. This hypothesis is supported by the fact that
the cranial ganglia resulting from the cranial mesenchyme area are
highly hypoplastic, and the branchial arches are underdeveloped in
AP-2 mutant animals at E 10.5 (9). Due to the hypoplastic
mesenchyme, the cranial neural tube would not get the physical support
needed resulting in insufficient bending of the tube at the lateral
hinge regions (31). This lack in bending finally leads to the neural
tube closure defect observed.
appear to have conserved
functional roles.
. Our data demonstrate that KLF-4 is expressed in
mutant mesenchymal cells at E 8.5 as well as fibroblasts lacking
AP-2. Furthermore in silico analysis of the promoter regions of KLF-4, Mtd, Stra13, and
mEFEMP-1 indicate that there are sites that fulfill the
criteria for AP-2 binding. However, further experiments will be
necessary to test directly the binding and transactivation of AP-2
on the promoter of KLF-4 as well as the other target genes reported.
mutants
can be tested by genetic complementation. The KLF-4 null mouse line had
been reported to complete embryonic development and die up to 1 week
after birth due to insufficient barrier function of the skin (38).
Generating a double mutant mouse deficient for AP-2 and KLF-4 will
serve to test this hypothesis.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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