(Received for publication, October 10, 1995; and in revised form, January 4, 1996)
From the
Retinoic acid (RA) and cyclic AMP analogs cause the
differentiation of F9 embryonic teratocarcinoma stem cells into
parietal endoderm, an epithelial cell of the early mouse embryo.
Laminin B1 is induced in this differentiation process, but is not
transcriptionally activated until 24-48 h after RA addition and
is not maximally induced until approximately 72 h. Cyclic AMP analogs
enhance this transcriptional activation. Although several DNase I
hypersensitive sites (DHSS) were observed in the LAMB1 5`-flanking DNA,
one of the sites, DHSS2, was detected only after 72 h of RA treatment.
Transient transfections have demonstrated that the DHSS2 region
functions as a ``late-acting RA-inducible enhancer,'' and
motifs in this enhancer contain the homeobox protein-binding site
TTATTAACA. Greater binding is observed at these sites by
electrophoretic mobility shift assay when cells are cultured with RA
and cyclic AMP analogs versus RA alone, and no binding is seen
in extracts from RA-treated F9 RAR cells
which lack RAR
mRNA and protein. Laminin B1 mRNA is not induced by
RA in the RAR
cells (Boylan, J. F.,
Lohnes, D., Taneja, R., Chambon, P., and Gudas, L. J.(1993) Proc.
Natl. Acad. Sci. U. S. A. 90, 9601-9605). Our data show that
these DNA regulatory elements contribute to the transcriptional
activation of the LAMB1 gene during the later stages of the
differentiation process.
Laminins are glycoproteins which are constituents of a
particular type of extracellular matrix called the basement membrane;
basement membranes are synthesized by epithelial, endothelial, nerve,
muscle, and other cell types. Laminins have a number of functions in
the processes of cell adhesion, cell migration, proliferation,
differentiation, neurite outgrowth, and tumor metastasis (for review,
see (2) ). Much information concerning the structure and
functions of laminins has come from the mouse Engelbreth-Holm Swarm
(EHS) tumor(3) . Three protein chains are constituents of EHS
laminin, Ae (M = 400,000), B1e (M
= 215,000), and B2e (M
= 205,000). These three polypeptide chains are assembled
into a cross-like structure with a molecular weight of approximately
950,000(4, 5) . Recently, additional laminin chains
related to Ae (Am; (6) and (7) ) or B1e (B1s; (8) ) have been reported. The human A chain homolog Am has also
been named merosin (6) and is the same as the chain found in
heart(7) . A specific deficiency in the laminin Am chain has
been associated with the dystrophic dy mutation in
mice(9, 10) . The dy mutation represents a
severe neuromuscular disease resembling human muscular dystrophy.
Furthermore, other laminin-related proteins have been
reported(11, 12, 13) . A human variant B2
chain named laminin B2t has been described(14) . An epithelial
specific laminin chain called laminin B1k has also been
reported(15) . Various heterotrimers can be assembled from the
different laminin subunits(16) . The nomenclature used is that
of Engel et al.(17) . Another nomenclature for the
protein chains has also been proposed recently; in this nomenclature,
the laminin B1 protein is called
1 and the gene is LAMB1(18) .
From sequencing clones from an EHS expression library, the cDNA sequences of the murine laminin B1e chain(19) , the B2e chain(20) , and the Ae chain (21) have been determined. The sequences of human laminin B1(22, 23) , B2 chain(14, 24) , and the A chain (25, 26) have also been determined.
During early mouse embryogenesis, mRNAs for the B1 and B2 chains of
laminin were detected from the 4-cell stage, while the A chain appeared
at the 16-cell stage(27, 28) . During the period of
mouse development in which major organs are forming, laminin was
detected in the basement membranes of epithelial cells(29) ,
during intestinal development(30) , and in ureteric buds and
nephrogenic vesicles during early prenatal kidney
development(31) . The conversion of mesenchyme to epithelium in
kidney is accompanied first by an increase in laminin B1 and B2
transcripts (31, 32) and is followed by an increase in
A chain expression(32) . The 1/
1 and
6/
1
integrin heterodimers have been shown to mediate cell attachment to
distinct sites on laminin (33) . The
6/
1 laminin
receptor is also regulated during development both by its level of
expression and via phosphorylation (34, 35, 36) .
The expression of laminin
has been studied in a number of model cell culture systems. In cultured
neuroepithelial cells, basic fibroblast growth factor enhances the
amount of laminin expressed at the protein level(37) . During
myogenic differentiation, the expression of several different laminin
chains is increased(38) . Retinoic acid, a member of the
vitamin A family of signaling molecules called retinoids, induces
laminin expression in cultured murine embryonic teratocarcinoma stem
cells such as the F9 cell line which is induced to differentiate into
parietal endoderm cells(39) . The levels of transcripts
encoding the laminin A, B1, and B2 polypeptide chains are increased in
F9 cells treated with RA ()as compared to undifferentiated
stem
cells(40, 41, 42, 43, 44, 45) .
The laminin B1 chain produced by RA-treated F9 cells appears to be
identical with the murine B1e chain as determined by DNA sequencing of
partial cDNA clones. (
)
Further studies by this laboratory
demonstrated that the RA-mediated regulation of the expression of the LAMB1 gene occurred primarily at the transcriptional
level(46, 47) . Cyclic AMP analogs were shown to
augment the transcriptional response of the cells to retinoic acid, but
cyclic AMP analogs alone did not enhance the rate of transcription of
this gene(44, 46, 47) . An RARE (retinoic
acid response element) was identified at position -477 to
-432 in the promoter region of the murine LAMB1 gene,
and this response element is recognized by retinoic acid receptors
(RARs)(48, 49) . Furthermore, the targeted disruption
of both copies of the RAR gene in F9 cells led to a defect in the
RA-associated activation of the LAMB1 gene(1) . This
suggested that the LAMB1 gene was a target gene regulated by
the RAR
. However, the late induction of the LAMB1 gene
and the observation that the RA-mediated induction was prevented by the
protein synthesis inhibitor cycloheximide (50) indicated that
other RA-inducible transcription factors were involved in the
regulation of the expression of the LAMB1 gene. In this
manuscript, we present a more detailed analysis of the regulation of
the LAMB1 gene in response to retinoic acid.
Figure 6: EMSAs using mutant oligonucleotides. A, radiolabeled wild type probe P2860 (lanes 1 and 2) and the radiolabeled mutant probe P2860M1 (lanes 3-7). 3 µg of extract from PYS-2 cells was used in lane 2. Nuclear extracts (5 µg) from F9 cells: stem cells (lane 3), RA for 24 h (lane 4), RACT for 24 h (lane 5), RA for 72 h (lane 6), and RACT for 72 h (lanes 1 and 7). B, wild type radiolabeled probe P2860 (lanes 1-5) and the radiolabeled mutant probe P2860M2 (lanes 6-10). Nuclear extracts from F9 cells: stem cells (lanes 1 and 6), RA for 24 h (lanes 2 and 7), RACT for 24 h (lanes 3 and 8), RA for 72 h (lanes 4 and 9), and RACT for 72 h (lanes 5 and 10). C, wild type radiolabeled probe P2821 (lanes 1-5) and the mutant probe radiolabeled P2821M1 (lanes 6-10). Nuclear extracts from F9 cells: stem cells (lanes 1 and 6), RA for 24 h (lanes 2 and 7); RACT for 24 h (lanes 3 and 8), RA for 72 h (lanes 4 and 9), and RACT for 72 h (lanes 5 and 10). D, the sequences of wild type and mutant oligonucleotides with the mutations underlined and a summary of binding sites for C1, C2 (solid box), and C3 (dotted box), respectively (sequences are written 5` to 3`). A + indicates the presence of a complex by EMSA; a - indicates the absence of a complex.
Figure 1:
Mapping DHSS of
the LAMB1 gene in F9 cells. A, the line diagram shows the LAMB1 gene with the positions of the ApaI (a), BamHI (b), HindIII (h), and PstI (p) sites in
the genomic DNA relative to the transcription initiation site and the
regions to which the DHSS maps marked. Probe SA is a 466-bp SacII/ApaI fragment covering from -353 to
+113 bp of murine LAMB1 gene (hatched box).
Probe HS is a 538-bp HindIII/SpeI fragment
covering from -3961 to -3423 bp of murine LAMB1 gene (open box). B, mapping of distal DHSS 5` to
the LAMB1 gene. Nuclei from F9 cells were treated with DNase I
(1, 3, and 10 units). DNA from DNase I-treated nuclei was digested with ApaI, and the Southern blot was hybridized with the probe SA
as described in A. The positions of DHSS are given. The RA
concentration was 1 10
M. This
experiment was performed three times, and similar results were obtained
in all three experiments.
To identify any DHSS closer to the initiation of transcription of the LAMB1 gene, DNA from DNase I-treated nuclei was digested with HindIII, analyzed by gel electrophoresis, and the resulting Southern blot was hybridized to a 538-bp HindIII/SpeI fragment containing -3961 to -3423 bp of mouse LAMB1 gene (Fig. 1A). Digestion of DNA in nuclei from untreated F9 stem cells resulted in a band of 3.7 kb (DHSS1) that maps to about -260 bp from the transcription initiation site. While this site, DHSS1, is located relatively close to the LAMB1 RARE(48) , this site was observed only in nuclei from stem cells and was not observed in DNA from nuclei of 24-h and 72-h RA-treated F9 cells (data not shown).
Overall, we have examined a total of 18 kb of genomic DNA at the 5` end of the murine LAMB1 gene. Three RA-sensitive DHSS were observed. DHSS1 is detected in F9 stem cells but is not observed after 24 h or 72 h of RA treatment. DHSS2 is observed only after 72 h of culture in the presence of RA, while DHSS3 is observed after both 24 h and 72 h of RA treatment. Because the DHSS2 was observed only at later times after RA addition (72 h), we concentrated on the further delineation of this site since its properties reflected the pattern of expression of the endogenous LAMB1 gene.
In order to define the regulatory elements in the DHSS 2 region of the murine LAMB1 gene in greater detail, the series of LAMB1 promoter/CAT constructs indicated in Fig. 2A were transfected into F9 cells, and this transfection was followed by treatment of the cells with RA for 24 h. For the 72-h time point, the cells were cultured in the presence of RA for 48 h, and then the cells were transiently transfected. This transfection was followed by further culture in the presence of RA for an additional 24 h. The results of these transient transfection assays indicated that all of the LAMB1 promoter/CAT constructs except the p0.13LAMCAT construct exhibited an increase in activity after 24 h of RA treatment (Fig. 2B). These results confirmed previous work by Vasios et al.(48, 49) which demonstrated that the p0.49LAMCAT construct which contains the RARE was RA-responsive by 24 h after RA addition.
Figure 2:
CAT activities of the reporter plasmid
containing the LAMB1 inserts. A, schematic
representation of the CAT reporter constructs. Numbering is relative to
the initiation site of transcription. The 3` end of LAMB1 insert is +113 bp. a, ApaI; b, BamHI; h, HindIII; and p, PstI. B, LAMCAT plasmids (10 µg) were
co-transfected with a murine RAR expression plasmid (1 µg),
p
AclacZ (5 µg), and pUC9 DNA to 20 µg total.
(Co-transfections with RAR
or RAR
gave similar results; data
not shown.) Transfections were normalized by assaying for
-galactosidase activity from the co-transfected p
AclacZ
construct. CAT activities of the constructs were measured and are
expressed relative to the F9 stem cell control (without RA addition).
The results shown are the averages of three independent transfection
experiments (mean ± S.D.).
Further increases in CAT activity were observed after 72 h of RA treatment for most of the LAMB1/CAT constructs (Fig. 2B). Deletion of a 569-bp fragment containing the DHSS2 from the 5` end of p3.4LAMCAT resulted in much less additional CAT activity at 72 h versus 24 h (Fig. 2B). Taken together, the DNase I hypersensitivity data indicating the location of DHSS2 at about -2.9 kb, and the results of these transient transfection assays are consistent with the hypothesis that the region of the LAMB1 promoter between -2.8 kb and -3.4 kb contains a DNA regulatory site which is more active at later times (72 h) after RA treatment than at 24 h.
Figure 3:
DNase I footprinting assays. A,
DNase I footprinting analysis with the nuclear extract from F9 cells
cultured in the presence of 1 10
M RA for 72 h. A probe spanning nucleotides -3136 to
-2735 (DraI/MseI fragment) was used. This probe
was prepared as described under ``Experimental Procedures.''
Increasing amounts of protein extract were used, from 1 µg to 40
µg. The lines indicate the footprints observed. The positions of
the footprints are also marked. This assay was performed four times
with different extracts; similar results were obtained. B, the
sequence of the LAMB1 gene from -2898 to -2787 is
shown. The footprinted sequences of individual binding elements are
indicated by the underlining. The footprinted sequence is
written 5` to 3`, with the nucleotide numbers shown on the left.
Figure 4:
EMSA analysis of the nuclear
protein-binding sites in F9 cells. EMSAs were performed with the
radiolabeled P2860, P2821 oligonucleotides (10 cpm/0.5 ng). Lane 1, radiolabeled probe alone. Lane 2, 5 µg of
the extract from F9 untreated stem cells. 5 µg of extract from F9
cells treated with RA for 24 h (lane 3), RACT for 24 h (lane 4), RA for 72 h (lane 5), and RACT for 72 h (lane 6) was used. The arrows indicate bands of
altered mobility representing specific protein-DNA complexes. A, probe P2860; B, probe P2821. The sequences of
probes P2860 and P2821 are shown in Fig. 6D. RA, 1
10
M; RACT is RA at 1
10
M plus dibutyryl cyclic AMP at 250
µM and theophylline at 500 µM. This
experiment was performed three times with different extracts; one
experiment is shown, but similar results were obtained in all three
experiments.
Analysis using the probe P2821 identified a single major retarded complex, C3 (Fig. 4B). The C3 complex is slightly more abundant in extracts from cells treated with RA for 24 h and in extracts treated with RACT for 24 h (Fig. 4B). Thus, while this C3 complex is present in nuclei from RA-treated cells, it is also present at significant levels in extracts from untreated F9 stem cells. From mutation studies described later in the manuscript (Fig. 6), the C3 complex observed with probe P2821 appears to be identical with the C3 complex observed with probe P2860.
Analysis with probe P2892 resulted in some very weak retarded complexes which appeared to be different from the complexes described above (data not shown). Analysis of these complexes was not pursued further.
Figure 5:
EMSA analysis of protein-binding sites
with corresponding unlabeled competitors. Labeled oligonucleotides
P2860 (A) and P2821 (B) with 4 µg of the nuclear
extract from F9 cells (1 10
M RA,
72-h treated) in the absence (lane 1) or in the presence (lanes 2-5) of the indicated (-fold) molar excess of
various unlabeled competitors: P2860 (lanes 2 and 3)
and P2821 (lanes 4 and 5).
The C3 complex formed with the P2860 probe was inhibited by a 100-fold molar excess of unlabeled P2821, whereas the C2 complex was not affected by a similar excess of P2821 (Fig. 5A). Reciprocal experiments showed that the C3 complex formed with the P2821 radiolabeled probe was inhibited by a 100-fold molar excess of unlabeled P2860 (Fig. 5B). These data suggest that the same protein is present in the complex C3 detected by both probes P2860 and P2821.
Mutation M1 in probe P2821 abolished C3 complex formation (Fig. 6C; Fig. 6D for the sequence of the radiolabeled P2821M1). From these data, we conclude that a TAAT sequence (complementary strand (ATTA)) in both P2860 and P2821 is involved in C3 complex formation.
In F9 wild type cells, the
expression of the parent plasmid pLBCAT2 was not RA-inducible, while pL
-3136/-2736TKCAT expression was RA-inducible (Fig. 7). In addition, the level of CAT activity of pL
-3136/-2736TKCAT could be enhanced by RACT as compared to
RA treatment (Fig. 7). The targeted disruption of the RAR
gene did not inhibit the RA-induced pL -3136/-2736TKCAT
expression (not shown). In contrast, the targeted disruption of the
RAR
gene abolished the RA-inducible pL
-3136/-2736TKCAT expression (Fig. 7). These data
show that the functional enhancer requires RAR
nuclear signaling.
This signaling is likely to be indirect since the DHSS2 region between
-3136 and -2736 does not appear to contain an RARE to which
RAR
could bind directly. Furthermore, in this experiment, the pL
-3136/-2736TKCAT construct displayed activity in the
absence of co-transfected RARs.
Figure 7:
The
effects of the targeted disruption of the RAR or RAR
gene on
the expression of the pL -3136/-2736TKCAT. The CAT
constructs (10 µg) were co-transfected with p
AclacZ (5 µg)
and pUC9 DNA (to 20 µg total) into F9 wild type, F9
RAR
, or F9 RAR
cells. No RAR expression vector was co-transfected. Cells were
then cultured in the presence of no addition, RA, CT, or RACT for 24 h,
followed by cell harvesting (RA, 1
10
M; C, 250 µM dibutyryl cyclic AMP; T, 500 µM theophylline). The experiments were
performed with 24-h drug treatment since a longer RACT treatment will
severely reduce the transfection efficiency. As a control, 4.4 ±
1.1 (mean ± S.D.; n = 4)-fold of induction was
observed when the pRAR
RARETKCAT was tested with 24-h RA treatment
(not shown). Transfections were normalized by assaying for
-galactosidase activity from the co-transfected p
AclacZ
construct. CAT activities of the constructs were measured and are
expressed relative to those of the corresponding plasmid without drug
addition. This experiment was performed twice with similar results; one
representative experiment is shown.
Extracts from these two cell lines
were then tested by EMSA to determine the effects of the loss of
RAR versus RAR
on the protein binding to regulatory
elements in this 400-bp DHSS2 region of DNA. The targeted disruption of
the RAR
gene had no effect on the behavior of the retarded
complexes (Fig. 8, compare lanes 2 and 3 with 4 and 5). In contrast, the targeted disruption of the
RAR
gene abolished the RA-associated C2 complex formation, but had
no effect on the behavior of the C1 or C3 complexes (Fig. 8, A and B). These data strongly suggest that
the C2 protein
DNA complex is of functional significance in the
positive regulation of the LAMB1 gene in response to RA since
this C2 complex does not form in extracts from
RAR
cells in which the LAMB1 gene is not activated by RA.
Figure 8:
EMSA analysis of the nuclear
protein-binding sites in RAR mutant cells. EMSA was performed with the
radiolabeled probe P2860 (A) and the radiolabeled probe P2821 (B). Lane 1, probe alone. Four µg of the nuclear
extracts were used from lanes 2-7: F9 wild type (lane 2), F9 wild type treated with RA for 72 h (lane
3), F9 RAR (lane 4), F9
RAR
treated with RA for 72 h (lane
5); F9 RAR
(lane 6), and
F9 RAR
treated with RA for 72 h (lane 7).
In order to
study the regulation of the LAMB1 gene, we searched for
changes in chromatin structure that occur when the gene is activated.
Our studies reveal that one of the DNase I hypersensitive sites, DHSS2,
was observed only at late times (72 h) after RA addition (Fig. 1B). Further, functional transient transfection
experiments have demonstrated that the DHSS2 region functions as an
enhancer ( Fig. 2and Fig. 7). Detailed analysis of this
enhancer by EMSA has led to the identification of several
proteinDNA complexes in this enhancer (Fig. 4, A and B). Among them, the protein complex C2 is most
intriguing because it is (a) found only in nuclear extracts of
RA-treated F9 cells and is more abundant at late times (72 h) than at
early times (24 h) (Fig. 4), (b) detected in extracts
from RA-treated F9 wild type cells and F9 RAR
cells, but not in RA-treated
RAR
cells (Fig. 8), and (c) detected at earlier times and in greater abundance in
extracts from RACT-treated cells versus RA-treated cells (Fig. 4A). Thus, our data are consistent with the
interpretation that the protein
DNA complex C2 contributes to the
activation of the LAMB1 gene during the differentiation
process. Our data also explain how a ``late''
cycloheximide-dependent RA-inducible gene such as LAMB1 can be
positively regulated in a delayed fashion in this differentiation model (Fig. 9).
Figure 9:
Model of LAMB1 gene regulation in
response to RA.(1, RA binds to RXRRAR
complex. (2), RA
RXR
RAR
complex binds directly
to LAMB1 RARE. (3), transcriptional activation of a
member of a homeodomain gene family (
) and homeodomain
transcription factor produced (requires approximately 24 h of RA
treatment). (4), cyclic AMP-dependent phosphorylation leads to
greater transcription or higher affinity binding of homeodomain protein
to elements at the DHSS2 region. (5), transcriptional
activation of LAMB1 gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43597[GenBank].