(Received for publication, August 7, 1996)
From the Verna and Marrs McLean Department of
Biochemistry and ** Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas 77030
Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.
Adenosine deaminase (ADA)1 is a purine catabolic enzyme that converts adenosine and deoxyadenosine to inosine and deoxyinosine, respectively (1). The enzyme is present throughout the evolutionary phyla, and the amino acid sequence is highly conserved from bacteria to humans (2, 3), suggesting it is a key enzyme in purine metabolism. In mice, the levels of ADA differ as much as 10,000-fold among various tissues with the highest levels occurring in the deciduum, chorioallantoic placenta, tongue, esophagus, forestomach, and proximal small intestine; intermediate levels are found in the thymus and low levels in other tissues (4). In addition, Ada expression is subject to developmental regulation at the maternal-fetal interface prenatally and in the gastrointestinal tract postnatally (4-8). Although the physiological role that ADA plays in these tissues is not clear, it is thought that ADA prevents accumulation of the cytotoxic metabolite deoxyadenosine, which interferes with deoxynucleotide metabolism and/or disrupts cellular transmethylation reactions (9-14). These disturbances result in apoptosis or disrupt cell differentiation.
Mounting evidence indicates that maintaining high levels of ADA in the placenta is essential for embryonic and fetal development. During placentation in mice, ADA appears at 7.5 days post coitum (dpc) in trophoblast giant cells surrounding the embryo and in diploid trophoblast precursor cells in the ectoplacental cone (6-8). The expression of Ada increases as the placenta develops. At 13.5 dpc when the placenta is mature, ADA appears in all trophoblast lineages, including trophoblast giant cells surrounding the gestation site, spongiotrophoblast cells in the junctional zone, and syncytial trophoblast cells in the labyrinth zone. By this time, greater than 95% of the ADA enzymatic activity found at the gestation site resides in the trophoblasts of the placenta (7, 22). Expression in the placenta persists until term, suggesting that ADA is functionally important in placental physiology. This importance is revealed by a series of mouse genetic experiments. ADA-deficient mice die perinatally of severe liver damage, brought on by profound disturbances in purine metabolism (15, 16). The concentrations of adenosine and deoxyadenosine increase 3- and 1000-fold, respectively, in ADA-deficient fetuses, while levels of inosine decrease. These metabolic disturbances are not evident until 12.5 dpc, coinciding with the lack of Ada expression in the placenta (15). By genetically restoring Ada expression specifically to the placenta, most of the purine metabolic disturbances in the fetus are prevented (17). Deoxyadenosine levels were lowered over 30-fold in ADA-deficient fetuses and severe fetal liver damage was prevented. As a result, ADA-deficient mice were rescued from the perinatal lethality (17, 18). Therefore, activating Ada gene expression in the placenta is critical for proper fetal development.
Studying Ada gene expression in the placenta will not only
enhance our appreciation of placental ADA, but also contribute to our
knowledge of trophoblast differentiation. ADA is an early marker of
trophoblast cell differentiation. Unlike other trophoblast-specific genes, whose expression is restricted to one cell type (19, 20, 21),
ADA appears in all trophoblast lineages. Thus, the Ada gene
may contain regulatory elements operable at all stages of trophoblast
differentiation and in all three cell types, representing a good system
to study trophoblast gene regulation. Starting with 6.4-kb 5-flanking
sequences, which direct chloramphenicol acetyltransferase (CAT) gene
expression to the placenta prenatally (and forestomach postnatally) in
transgenic mice (22), we have localized the placenta-specific
regulatory element to a 770-bp fragment 5.4 kb upstream of the
Ada transcription initiation site using transgenic mice as
an assay system. The speed and the efficiency of this analysis were
increased by examining expression of transgenes in placentas 14 days
following zygote microinjection. In situ hybridization
showed CAT reporter gene expression in the same cell types that
expressed ADA. Sequence analysis and DNase I footprinting experiments
revealed both general and placenta-specific protein binding sites
within this region. Deletions and site-specific mutations of potential
protein binding sites within this 770-bp fragment resulted in loss of
placenta-specific expression, suggesting that multiple factors function
together to ensure proper Ada expression in the
placenta.
The 6.4CAT transgene is described by
Winston et al. (22) as the construct ADACAT. This construct
was subcloned into the BamHI site of Bluescript
KS+ II vector (Stratagene). Deletions were prepared by
appropriate restriction digest of this parental plasmid. In short,
5.5CAT, 2.7CAT, and PCAT were generated by ClaI,
NcoI, and XbaI digestion of 6.4CAT. 3.1PCAT,
2.0PCAT, FP3PCAT, 1.8PCAT, 1.3PCAT, and 0.77PCAT were generated by
ligation of PCAT to various restriction fragment from the 6.4-kb
Ada flanking sequence. To generate mutant constructs, a
770-bp XbaI-PstI fragment was subcloned in
Bluescript KS+ II vector. The 5
deletion was generated
according to the instructions of the Erase-a-Base system (Promega). The
FP1 deletion and GATA mutations were generated using the
Muta-Gene phagemid in vitro mutagenesis system, version
2 (Bio-Rad). The mutagenic oligonucleotides, FP1
(GTTGAAGAGGAAACAGCGGTACCACGGGTCATCATGAGTTTTG) and GATAm
(GGAGGAACATAAACTTAATCATCTTTGTCATC), were synthesized by the Institute
of Molecular Genetics Nucleic Acids Core Laboratory, Baylor College of
Medicine. The mutations were confirmed by nucleotide sequencing prior
to ligation to PCAT as 5
240PCAT, FP1
PCAT, and GATAmPCAT. PCAT
contained murine Ada sequences from the XbaI site
at
750 bp to the NcoI site at +90 bp ligated to CAT coding
region followed by the SV40 small t intron and late poly(A) signals.
Prior to microinjection, prokaryotic vector sequences were removed by
restriction digestion.
Each transgene was isolated by agarose gel electrophoresis and was purified by the QIAEX II procedure (QIAGEN Inc.). DNA was resuspended at 3 ng/µl in 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA and was microinjected into the male pronucleus of fertilized FVB/N oocytes (23). Fourteen days following DNA injection, gestation sites were separated into embryos and placentas for CAT assays, and genomic DNA was prepared from the fetal yolk sacs for genotyping. When necessary, lines were established by mating with ICR mice, and DNA was obtained from tail biopsies of 4-week-old offspring.
DNA HybridizationsTransgenics were identified and transgene copy number was determined by DNA dot blot as described (22). For Southern blots, DNA was transferred to Zeta-Probe membrane (Bio-Rad) and hybridization was done as described (24).
Tissue Extracts and CAT Assays-Tissue extracts were prepared and CAT assays were performed as described (22). Typically, the placenta and embryo extracts were incubated in a solution containing 385 mM Tris-HCl, pH 7.8, 33 mM [14C]chloramphenicol, and 1 mM unlabeled acetyl-CoA at 37 °C, and the reactions were stopped by adding ethyl acetate. Substrate and acetylated products were resolved by thin layer chromatography (Sigma) and visualized by autoradiography. The specific radioactivities of the substrate and the products were measured using a Betascope 603 Blot Analyzer (Betagen).
In Situ HybridizationsEmbryos and placentas were fixed in
4% paraformaldehyde, phosphate-buffered saline overnight and processed
for in situ hybridization as described (25). RNA probes were
labeled with [-35S]UTP (1000 Ci/mmol, Amersham). The
ADA probes were generated from a 310 bp fragment from the 5
end of the
cDNA. The CAT probes were generated from the first 250 bp of the
coding sequence. Samples were hybridized overnight at 60 °C and were
treated as described (25). Slides were dipped in Kodak NTB-2 emulsion
and exposed for 5 days. Sections were viewed and photographed with a
Leitz Diaplan microscope (26). The micrographs shown in Fig. 2 are double exposures with the red representing hybridization
signal using dark-field optics with a red filter, and the
blue representing nuclei stained with Hoechst 33258, viewed
with epifluorescence optics.
DNA Sequencing
The 770-bp XbaI to PstI fragment was subcloned into Bluescript KS+ II vector for sequencing by the dideoxy method utilizing the Sequenase 2.0 kit (U. S. Biochemical Corp.) following manufacturer's instructions. Each strand was sequenced by a series of oligonucleotides that were synthesized by the Institute of Molecular Genetics Nucleic Acids Core Laboratory, Baylor College of Medicine. Nucleotide sequence comparisons were performed with the GAP program, and transcription factor consensus sequences were sought using the Findpatterns program both from the Genetics Computer Group program, University of Wisconsin (27).
Nuclear Extracts and DNase I FootprintingNuclei were
prepared from mid-gestation murine ICR placentas (12.5-15.5 dpc) or
livers removed from 8-12-week-old murine ICR females as described
(28), and nuclear extracts were prepared by the procedure of Dignam
et al. (29). For footprinting, the 770-bp Ada
placental regulatory element was subdivided into a XbaI to
SacI 510-bp fragment and a SacI to
PstI 258-bp fragment, and each fragment was subcloned into
Bluescript KS+ II vector. The plasmid was linearized with
an enzyme that leaves a 5 overhang and calf intestinal phosphatase
(alkaline)-treated. The fragment was released by a second digest
utilizing an enzyme that produces a 3
overhang and was gel-purified.
Fragment was end-labeled by polynucleotide kinase (Boehringer Mannheim)
utilizing [
-32P]ATP (3000 Ci/mmol). The radiolabeled
fragment was incubated with either placenta or liver nuclear extract
for 45 min at room temperature in Dignam D buffer containing 2 µg of
poly(dI-dC) in a total volume of 50 µl. 0.8 units of DNase I were
added for 2 min at room temperature. Reactions were stopped, and DNA
samples were resolved on either 6% (500-bp fragment) or 8% (250-bp
fragment) polyacrylamide urea sequencing gels run in 1 × TBE.
We have shown previously that a placental regulatory element lies within a 6.4-kb region immediately upstream of the murine Ada gene (22). We sought to identify the placenta-specific regulatory element by deletion analysis in transgenic mice. Transgenic mice were used because this approach provides a physiological assay system in which the complete array of necessary regulatory elements could be defined in a developmental context. To increase the pace of the analysis, we chose to study founder mice instead of generating transgenic lines since the same features of CAT expression were observed in both assays (Table I). Of nine 6.4CAT transgenic lines, seven showed high levels of CAT activity in the placentas and undetectable CAT activity in the adjoining embryos. Two lines with a single copy of transgene displayed very low levels of CAT activity in both placentas and embryos. All six 6.4CAT founders showed high levels of placenta-specific CAT expression. The level of CAT expression varied considerably among the 6.4CAT transgenic lines and founders and did not correlate with copy number. Thus, the data presented in Table I indicated that transgenic founders worked as well as the lines to define the Ada placenta-specific regulatory element.
|
We next tested various 6.4CAT deletion constructs for their ability to
target CAT expression in the placentas of founder mice 14.5 dpc (Fig.
1). Three 5 deletion constructs from 5.5 to 0.8 kb did
not show CAT activity in the placentas, suggesting that the placental
regulatory element resided at the 5
part of the 6.4-kb fragment. The
smallest construct, PCAT, which contained the Ada promoter,
was used as a basal transcription unit to identify the upstream
placental regulatory element. Five additional deletion constructs
narrowed the placental regulatory element to a 770-bp XbaI
to PstI fragment located about 5.4 kb upstream of the
Ada transcription initiation site, which in combination with
PCAT was able to direct high levels of CAT expression in the placentas of transgenic mice. CAT activities in the adjoining embryos were undetected in most transgenic mice. In some cases (two 2.0PCAT mice and
one 1.8PCAT mouse), embryos displayed low levels of CAT activity but
were more than 10-fold less than that in the adjoining placentas.
Additionally, one 2.0PCAT and two 0.7PCAT founders did not show any CAT
activity in either placentas or embryos, presumably due to the effects
of the integration sites. These data indicated that the 770-bp region
contained the Ada placenta-specific genetic regulatory
element.
The Ada Placental Regulatory Element Directs Reporter Gene Expression to the Same Cell Types as the Endogenous Ada Gene with Appropriate Developmental Timing
To confirm that CAT gene expression occurred in the same cell types as endogenous ADA, the cellular localization of CAT transcripts from the 2.0PCAT transgene (Fig. 1B) were identified by in situ hybridization. A transgenic male containing approximately 18 copies of the 2.0PCAT transgene was bred to non-transgenic ICR females. Placentas and embryos were isolated at 9.5 and 13.5 dpc. Day 13.5 tissues were genotyped from DNA obtained from extraembryonic membranes while 9.5-dpc transgenics were identified by the presence of CAT transcripts. Tissue sections were hybridized to either CAT or ADA sense or antisense radiolabeled RNA probes. Both CAT and ADA antisense probes produced strong signals in trophoblast cells (Fig. 2). At 9.5 dpc (Fig. 2, compare panels A and B), ADA and CAT transcripts were most abundant in the spongiotrophoblasts of the developing junctional zone. Hybridization was also observed in diploid precursors. In higher magnification of panels A and B (Fig. 2, C and D), both ADA and CAT mRNA was observed in the secondary giant trophoblasts. At 13.5 dpc (Fig. 2, compare panels E and F), the strongest signals for each gene occurred in the spongiotrophoblasts of the junctional zone. Both CAT and ADA mRNA were also visible in the syncytial trophoblasts and the secondary giant trophoblasts in the labyrinth zone. Neither ADA nor CAT mRNA was detected by this procedure in embryo sections (data not shown), although very low levels of ADA enzyme activity is detected in embryos (6). These results demonstrate that the placental signal within the 2.0-kb BamHI to EagI fragment directs CAT expression to the ADA expressing trophoblasts in the placenta. CAT expression observed during placental formation suggested that this placental signal activates CAT expression at the proper time in development.
The Ada Placental Regulatory Element Fails to Direct Expression to the ForestomachSince the original 6.4CAT construct also directs
expression to the forestomach in adult mice (22), we wished to
determine whether this placental regulatory element can function in the forestomach of adult mice. To address this question, a male founder harboring the 2.0PCAT transgene was mated to several non-transgenic ICR
females. One pregnant female was sacrificed at 14.5 dpc to obtain
placentas. The remaining females were allowed to go to term, and the
resulting litters were sacrificed at 5 weeks. CAT activity was measured
in extracts prepared from various tissues. CAT activity was apparent in
transgenic placentas, but was not detected in the forestomach or other
tissues of adult transgenic F1 mice (Fig. 3). The 2.0-kb
BamHI to EagI fragment containing the
Ada placental regulatory element appeared to lack element(s) essential for forestomach expression. This result was confirmed with a
second 2.0PCAT line. Thus, the Ada placental regulatory element appeared to have only the capability of directing enhanced reporter gene expression to the placentas in transgenic mice.
The Ada Placental Regulatory Element Contains Sequence Motifs Homologous to Transcription Factor Binding Sites Found in Other Placenta-specific Genes
To identify potentially important
regulatory sequences within the Ada placental regulatory
element, its sequence was determined and analyzed for homology with
regulatory regions of other genes expressed in trophoblasts (Fig.
4). A significant stretch of homology was found with the
human placental lactogen (hPL) gene enhancer (30) (Fig. 4B). With
exception of a 12-bp gap in the middle, the sequence between
nucleotides 57 and 103 on the Ada placental regulatory
element showed 77% identity with footprinting region 3 in the hPL
enhancer. Interestingly, the gap matched well with footprinting region
4 of the hPL enhancer (66% identity). However, no significant stretch
of homology was found with the 4311 spongiotrophoblast enhancer region
(31).
The 770-bp Ada placental regulatory element was rich in transcription factor binding sites including some playing a role in trophoblast-specific gene expression (Fig. 4A). Two sequence motifs similar to the GATA consensus sequence (WGATAR) (32) were observed at nucleotides 376 and 390 and two consensus cAMP response elements (33) were found at positions 26 and 598, respectively (Fig. 4). One trophoblast-specific element (TSE) consensus sequence (CCNNNGGG) (34) was at 336 and five basic helix-loop-helix (bHLH) sequence motifs (CANNTG) (35) representing potential binding sites for either Mash-2 (39) or Hxt (43) were also identified. Thus, this 770-bp placental regulatory element contained sequence motifs of potential functional importance.
A Placenta-specific Protein-binding Region Was Detected within the 770-bp Ada Placental Regulatory ElementTo directly probe the
protein binding sites in the 770-bp Ada placental regulatory
element, DNase I footprinting experiments were performed in the
presence of either placenta nuclear extracts or liver nuclear extracts.
The Ada placental regulatory element was divided into a
510-bp XbaI to SacI 5 fragment and a 258-bp SacI to PstI 3
fragment for footprinting. A
single footprint was detected on the 5
fragment that covered
nucleotides 241-270 (FP1, Fig. 5, A and
B) in placenta but not liver nuclear extracts. This
footprint contained two sequences similar to the TSE consensus sequence
CCNNNGGG (34), suggesting FP1 may represent an essential, placenta-specific regulatory region. The 3
fragment contained two
footprints: FP2 (nucleotides 641-670) and FP3 (nucleotides 731-750);
each one appeared in both liver and placenta extracts (Fig. 5,
C and D). Thus the Ada placental
regulatory element is capable of binding both placenta-specific and
potentially general factors.
Different Sequence Motifs in the Ada Placental Regulatory Element Contribute to Ada Placenta-specific Expression
Sequence analysis
and DNase I footprinting experiments indicated several sequence motifs
in the Ada placental regulatory element with potential
importance. The first 240 bp of the Ada placental regulatory
element contained sequence homologous to the human placental lactogen
enhancer and (four) consensus bHLH binding sites (Fig. 4). However, we
were unable to detect any protein binding in this region by the DNase I
footprinting experiments (Fig. 5). To examine the importance of this
region, the 5 240-bp sequence of 0.7PCAT was deleted and its ability
to direct placental expression was tested in transgenic mice (Fig.
6). Five founder mice were generated containing
different copies of the 5
240PCAT transgene. No CAT activity was
detected in either placentas or embryos of two founders, and low
placenta-specific CAT activity appeared in another two mice. In one
case, low levels of CAT activity were detected in the embryo but not in
the placenta. These data indicated that the 5
240-bp fragment is
required to confer reliable placenta-specific expression in transgenic
mice.
A strong placenta-specific protein binding region (FP1) detected in the
Ada placental regulatory element implied its functional importance. To determine whether FP1 was a critical part of the Ada placental regulatory element, 18 bp of the FP1 region in
the Ada placental regulatory element were deleted and the
mutant construct FP1PCAT was tested in transgenic mice. The
resulting four founders with copy numbers ranging from 3 to 70 did not
display CAT activity in either placentas or embryos (Fig. 6). These
data strongly indicated that the placenta-specific protein/DNA
interaction in the FP1 region is essential for Ada
expression in the placenta.
Two consensus GATA sites were identified 7 bp apart in the
Ada placental regulatory element. GATA factors were reported
to be involved in regulation of human chorionic gonadotropin subunit gene and mouse placental lactogen gene expression in the
trophoblast cells (36, 37). To examine whether GATA motifs are required for Ada expression in the placenta, G
C point mutations
were introduced into both GATA sites in the Ada placental
regulatory element, and the effect of these mutations on placental
expression was tested in transgenic mice (Fig. 6). Of four transgenic
mice, two showed placenta-specific CAT expression compared to that in the embryos but the level of expression was low to barely detectable in
the placenta; one showed very low but detectable CAT activity in both
placenta and embryo; and one did not show any CAT activity. These
results suggested that one or both GATA motifs are required for
enhanced Ada expression in the placenta.
The Ada placental regulatory element contained motifs not
only for placenta-specific factors, but for potential ubiquitous factors as well. One region, FP3, was tested for its contribution to
Ada expression in the placenta. The FP3 deletion construct FP3PCAT, which had a 34-bp deletion from the 3
end of the
Ada placental regulatory element, was unable to direct CAT
expression in five transgenic mice (Fig. 6). Therefore, the sequence
motifs in FP3, which bind proteins present in both livers and
placentas, are required for placenta-specific expression of
Ada.
In summary, the mutational data demonstrated that both placenta-specific motifs (FP1), and potential ubiquitous motifs (FP3), as well as GATA motifs and possible bHLH motifs, are required for Ada expression in the placenta.
As part of our effort to understand the physiological importance of placental ADA in prenatal development, we wish to identify signaling pathways that govern the temporal and cellular expression of the murine Ada gene during placental development. Our approach involved the use of a relatively convenient and rapid transgenic assay in which the expression of ADA/CAT reporter genes was determined in transgenic placentas 14 days following zygote microinjection. The data here represent the identification and detailed analysis of a 770-bp sequence located approximately 5.4 kb upstream of the transcription initiation site of the murine Ada gene, which, in combination with an 800-bp region containing the Ada promoter, is sufficient to target reporter gene expression to the placentas in transgenic mice. In the absence of the placental regulatory element, no detectable CAT activity is produced from the Ada promoter in either the placenta or embryo, and the combination of the two regions directs expression only to the placenta and not to other tissues. Because the Ada promoter can be highly activated in a variety of unrelated cell types (trophoblasts, gastrointestinal epithelium, uterine decidual cells, and thymocytes), we believe that trophoblast specificity lies with the 770-bp upstream regulatory element. Consistent with this view are unpublished data indicating that the 770-bp fragment can activate heterologous promoters in a placenta-specific manner.2 The Ada placental regulatory element is capable of directing reporter gene expression in all trophoblast lineages and in a manner that coincides with the appearance of endogenous ADA transcripts in this cell lineage (6-8). These results indicate that the Ada placental regulatory element contains regulatory motifs capable of functioning in all three murine trophoblast lineages and at the correct developmental time.
DNase I footprinting experiments determined that protein(s) present in
placenta nuclear extracts bind to the 770-bp fragment at three places,
termed FP1, FP2, and FP3. FP1 binds proteins present in nuclear
extracts from placenta but not liver, suggesting that the protein/DNA
interaction is placenta-specific. Deletion of FP1 resulted in loss of
placenta-specific expression, indicating that sequence motifs within
this region are required for placenta-specific expression. Inspection
of the FP1 sequence revealed that it contains two motifs similar to the
consensus sequence for the trophoblast-specific element-binding
protein, TSEB (34). This protein is present in the human
choriocarcinoma cell line JEG-3 and binds the TSE that is part of the
cis-regulatory sequence required for the enhanced expression
of the human gonadotropin and
subunit genes and the aromatase
gene in that cell type (34, 38). Although mouse placenta does not
produce gonadotropin, it is possible that a murine equivalent of TSEB
is participating in the regulation of the Ada gene in the
mouse placenta. The other footprinting regions, FP2 and FP3, bind
proteins present in both liver and placenta nuclear extracts, and they
presumably involve widely distributed proteins that may play supporting
roles in achieving tissue-specific gene expression. The importance of
FP3 in the Ada placental regulatory element was revealed by
a deletion mutant that removed 34 bp, including a portion of FP3, from
the 3
end of the Ada placental regulatory element.
Transgenic animals carrying this deleted transgene failed to show
placenta-specific expression. This finding suggests that in addition to
placenta-specific factors associated with FP1, other more generally
expressed factors associated with FP3 are required for
placenta-specific expression. These features of the Ada
placental regulatory element are consistent with the concept that cell
specificity is achieved in part by tissue-specific regulatory elements
and in part by regulatory elements that bind more widely distributed
proteins.
Although the footprinted regions represent good candidates for
functional components of the placental regulatory element, the lack of
observable footprints over other regions does not exclude the
possibility that other factors of functional importance may bind
in vivo. Moreover, since the entire placenta was used to
prepare nuclear extracts, regulatory proteins present in small populations of cells, such as the giant cells, may not have been sufficiently concentrated to be detected in this assay. In view of
these potential limitations of footprint assays, the sequence of the
Ada placental regulatory element was closely examined to identify sequence motifs common to other trophoblast-specific genes. At
the 5 end, the sequence from 57 to 103 bp shows high homology to the
human placental lactogen enhancer footprinting regions DF3 and DF4. DF3
and DF4 bind nuclear proteins and mediate a synergistic
trophoblast-specific enhancer activity in JEG-3 cells (30). The
homology between the human placental lactogen enhancer and the
Ada placental regulatory element suggests that similar
factors may be involved in the placenta-specific expression of each
gene. The potential importance of the human placental lactogen homology
region is underscored by the fact that it lies within an essential
240-bp region at the 5
end of the Ada placental regulatory
element. The Ada placental regulatory element also contains
five binding sites for bHLH proteins (35). Two bHLH sites lie within
the human placental lactogen homology region and may represent critical
components of the conserved sequence. Two bHLH factors, Mash-2 and Hxt,
are specifically implicated in gene regulation during trophoblast
development (39-44). Mash-2 is found in both trophoblast precursors
and spongiotrophoblasts and is essential for spongiotrophoblast
development (39-41). Hxt is present in early trophoblasts and in
differentiated giant cells and can induce commitment of blastomeres to
trophoblasts (42-44). Although specific target genes of Mash-2 and Hxt
have not been identified, available evidence favors an important role
for these proteins in regulation of trophoblast gene expression. A
potential role for bHLH factors in the regulation of Ada
gene expression is consistent with the fact that four of the five bHLH
sites found in the Ada placental regulatory element are
present within the critical 240-bp fragment at the 5
end. Additional
experiments are required to specifically assess the importance of bHLH
proteins in the regulation of Ada gene expression in
trophoblasts.
There are two centrally located GATA sites in the Ada
placental regulatory element. GATA factors are zinc finger
transcription factors that play a pivotal role in hematopoiesis and
erythroid gene expression (45-51). Two members of this gene family,
GATA-2 and GATA-3, are found in trophoblasts of the placenta (37, 52). The GATA motif is part of the trophoblast-specific enhancer that regulates the human chorionic gonadotropin subunit gene expression in JEG-3 cells (36). GATA factors are also involved in
trophoblast-specific expression of mouse placental lactogen I gene in
rat Rcho-1 cells (37). The possible involvement of GATA factors in
regulating Ada expression in the placenta was investigated
by introducing point mutations into each of the GATA motifs. Lack of
CAT activity in placentas harboring such mutant transgenes suggests
that GATA factors are required to activate the Ada gene in
the placenta. GATA factors may be essential for activation of the
Ada gene expression in the ectoplacental cone and/or
maintaining Ada gene expression in all branches of the
trophoblast population.
The evidence that Ada placental regulatory element is composed of a series of distinct genetic regulatory motifs suggests that a combination of multiple transcription factors are required for Ada expression in the mature placenta. These potential factors include bHLH factors, GATA factors, TSEB, and other ubiquitously expressed factors. Identification of these factors and characterization of their function in Ada expression are currently under way. It is possible that different set of transcription factors are required for Ada expression at different stages during placenta development. Some of these factors may be involved in activation of Ada in the ectoplacental cone, some may participate in maintaining high levels of Ada expression in one or multiple trophoblast lineages.
In addition to placental trophoblasts, the murine Ada gene
is also expressed at high levels in several diverse cell types (4-8):
the keratinized epithelium of the tongue, esophagus, and forestomach;
the absorptive epithelium of the small intestine; and the decidual
cells of uterine stroma (4-8). Ada is expressed at
intermediate levels in the thymus and low levels in most other tissues
(2, 4). The results presented here indicate that the Ada
placental regulatory element is capable of directing enhanced expression only to the placenta, suggesting that it is functionally independent from other tissue-specific regulatory elements. Since the
original 6.4-kb 5-flanking region also directs reporter gene expression to the forestomach postnatally (22), it appears that the
placenta and forestomach regulatory elements are distinct. In this
regard, recent experiments have identified a distinct and completely
independent regulatory region located approximately 1 kb downstream of
the placental regulatory element capable of directing expression
specifically to the forestomach.3 A
regulatory region in intron 1 functions to direct enhanced CAT
expression to the thymus (53-56). This same region also produces low
levels of expression in most tissues of transgenic mice, suggesting that it contains regulatory elements that contribute to the ubiquitous expression of the Ada gene (56). Recent results with the
intron 1 regulatory region indicate that the ubiquitous elements do not require the thymus-specific elements to function (56). Thus, we have
identified individual regulatory elements for enhanced expression in
the placenta, forestomach, and thymus, as well as a regulatory region
required for ubiquitous expression. Regulatory elements not yet
identified include those for the tongue, esophagus, small intestine,
and deciduum. We speculate that separate regulatory elements will be
required for each of these remaining tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U73185[GenBank].