(Received for publication, February 19, 1997, and in revised form, May 5, 1997)
From the Laboratory of Molecular Cardiology, NHLBI,
National Institutes of Health, Bethesda, Maryland 20892 and the
¶ Department of Biochemistry and Molecular Biology, The University
of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
NK-4, also called msh2 and tinman, encodes a homeodomain transcription factor that is required for the development of the dorsal mesoderm and its derivatives in the Drosophila embryo. Genetic analyses indicate that NK-4 resides downstream of the mesodermal determinant twist, which encodes a basic helix-loop-helix-type transcription factor. However, the regulation of NK-4 by twist remains poorly understood. Using expression assays in cultured cells and transgenic flies, we show that two distinct clusters of E-box regulatory sequences, present upstream of the NK-4 gene, mediate NK-4 expression in the visceral mesoderm. These elements are conserved between the Drosophila melanogaster and Drosophila virilis NK-4 genes and serve as binding sites for Twist (E1 cluster) and NK-4 (E2 cluster) proteins. In cultured cells, Twist and NK-4 binding results in activation of NK-4 gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected, and both elements are required for NK-4 activation in cells of the visceral mesoderm and also for NK-4 repression in cells of the somatic musculature. These results demonstrate that NK-4 is a direct transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the idea that twist and NK-4 function in the subdivision of the mesoderm during Drosophila embryogenesis.
In Drosophila, the mesoderm develops from cells in the ventral-most part of the embryo at the cellular blastoderm stage (reviewed in Ref. 1). After gastrulation, the mesoderm is further subdivided into three different mesodermal layers including the somatic mesoderm, visceral mesoderm, and progenitors of the heart. Although the mechanism of mesoderm partitioning into more specialized cells is unclear, it is generally believed that a cascade of transcriptional regulators and inductive signals from other germ layers are involved (2-9).
Previously, it was shown that twist, which encodes a basic helix-loop-helix (bHLH)1 transcription factor, is essential for the early establishment of the mesoderm (10, 11). twist is initially activated by dorsal in the presumptive mesoderm of the cellular blastoderm embryo (12-16), where the highest concentration of the dorsal morphogen exists. Subsequently, Twist is distributed in a graded manner where it regulates downstream genes (17, 18). Possible targets of twist include msh-2 (19), PS2 (18), Zfh-1 (20, 21), DFR1 (22), and D-mef2 (23-26), since mesodermal expression of these genes is disrupted in twist mutant embryos. Recently, it was demonstrated that Twist also functions in the subdivision of the mesoderm, presumably selecting different targets in mesodermal derivatives depending on its concentration (27). Although it was suggested that Twist is required for the activation of those mesodermally expressed genes, little evidence of their direct activation by Twist in specialized mesodermal cells has been published to date.
NK-4, also named msh-2 and tinman, is a mesodermal gene that belongs to the cluster of homeobox genes (lbe, lbl (nkch4), NK-4 (msh-2, tinman), NK-3 (bagpipe), 93Bal, and NK-1 (S59)) that are located in the 93D/E region of Drosophila chromosome III (28-34). NK-4 is initially expressed in the presumptive mesoderm at the cellular blastoderm stage shortly after twist, and it continues to be expressed in all mesodermal cells during germband elongation (19).2 Eventually, its expression is restricted to dorsal mesodermal cells including precursor cells of the heart (19, 30). As revealed by the analysis of the tinman mutant embryos, the gene is required for visceral mesodermal cell differentiation and development of the dorsal vessel that is functionally comparable to the mammalian heart (30, 31). Recently, Gajewski and co-workers (35) showed that NK-4 activates the D-mef2 transcription factor gene in cardial cells during heart morphogenesis. These studies demonstrated that D-mef2 resides directly downstream of NK-4 in the genetic hierarchy controlling heart formation in Drosophila. NK-4-related genes have been identified in vertebrates, and their patterns of expression were shown to be confined to the developing heart and gut tissue (36-42). Furthermore, targeted disruption of the mouse Nkx-2.5 gene results in abnormal heart formation during embryogenesis, suggesting that Nkx-2.5 is essential for normal heart morphogenesis (43). These results raise the possibility that the genetic pathways required for heart morphogenesis may be conserved among different species (44-46).
As an initial effort to understand the molecular mechanism by which NK-4 functions in specifying the regional subdivision of mesoderm and heart development, we have investigated the transcriptional control of NK-4 in cultured cells and transgenic flies. We show that NK-4 is a direct target for Twist in visceral mesodermal cells. Moreover, we show that this regulation is mediated by two distinct clusters of E-box regulatory elements and involves autoregulation by the NK-4 protein.
A Drosophila
melanogaster genomic DNA library (cosmid library,
CLONTECH) was screened with a 2.3-kb
EcoRI-BamHI fragment corresponding to the NK-4
first exon and part of the first intron (clone 9 from Ref. 28) to
isolate clones containing the 5 upstream region of the NK-4 promoter.
One of the cosmid clones obtained (CL 3; 23-kb insert) was used for
subcloning and restriction mapping. For sequencing, two subclones
(1.62-kb EcoRI insert and 2.3-kb EcoRI-BamHI insert) were serially deleted with
ExoIII-S1 nuclease (Promega), and serial deletion mutant plasmids were
used as template DNA for DNA sequencing with Sequenase (U. S. Biochemical Corp.). By aligning the overlapping sequences, the total
3.92-kb sequence was obtained. For cloning of the twist
cDNA, we synthesized oligonucleotides (primer I, 5
CTGGAATTCCACAAATTCTAACGTGAAGAAGG 3
; primer II, 5
GGCTTAGACATCTTAGAATCATCT 3
) based on the published sequence (11) and
used them for polymerase chain reaction (PCR) (35 cycles of 1 min at
95 °C, 1 min at 55 °C, 2 min at 72 °C) with
Drosophila embryonic (3-12 h) cDNA templates. Amplified
DNA was digested with EcoRI and subcloned into the
pBluescript KSII vector (Stratagene). Cloned cDNAs (pKS-Twist,
1.6-kb EcoRI insert from nucleotide 108 to 1823 without
intron) were confirmed by sequencing and used for construction of the
expression vectors. For the cloning of the DvNK-4 homeobox
gene, an lEXlox Drosophila virilis genomic DNA
library (Novagen) was screened with a 32P-labeled-NK-4
cDNA.
A 0.97-kb EcoRI
twist cDNA fragment (encoding amino acids 169-490) was
excised from pGBT-TwiQ3 and inserted into
the EcoRI site of pGEX5X-1 (Pharmacia Biotech Inc.). For the
GST:NK4 fusion protein, plasmid
pGAL4:NK4A24 was digested with
EcoRI, and the 0.73-kb DNA fragment (encoding amino acids
188-416) was eluted from a gel and subcloned into the EcoRI
site of pGEX5X-1. Resulting plasmids (pGEX:TwiQ and pGEX:NK4-A2) were
transformed into Escherichia coli BL21 (Novagen). Cells were
grown at 37 °C (up to A600 = 0.7), and
expression of fusion proteins was induced by
isopropyl-1-thio--D-galactopyranoside treatment (final
concentration, 0.2 mM). Fusion proteins were prepared
according to the method of Ip et al. (47), with three cycles
of freezing and thawing in the lysis buffer (25 mM HEPES (pH 7.5), 20 mM KCl, 2.5 mM EDTA, 1% Triton
X-100, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml of pepstatin, 1 µg/ml of
leupeptin) followed by affinity purification with glutathione-Sepharose beads (Pharmacia). Fusion proteins were eluted from the beads, dialyzed
against 300 volumes of buffer containing 25 mM HEPES (pH
7.5), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 6 mM MgCl2, and 10%
glycerol, and the protein was stored at
70 °C.
For constructions of F-series
reporter plasmids, DNA fragments (F23, 1186 to
887; F33,
1036 to
737; F43,
888 to
587; F53,
738 to
438; F3,
888 to
737)
containing various 5
upstream regions of NK-4 were
amplified by PCR (30 cycles of 1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C) with specific primers. For the constructions of the
F23E1m and F33E2m, P1E1mE2m plasmid DNA (see below) was used for PCR as
template. Primers (27-mer including restriction site) for the 5
to 3
direction have a HindIII site in addition to the
corresponding sequence, and those for the 3
to 5
direction have a
BamHI site at the end. Amplified DNA fragments were cut with
HindIII and BamHI and subcloned into the pBLCAT2 (48). To construct the P-series reporters (P1, P3, and P5) containing the NK-4 promoter and upstream region, a common primer for
the 3
to 5
direction containing the SalI restriction site
at the end (primer 750 from
137 to
154, 5
CTGGTCGACAACCGTTAGCGCAACCGT 3
) and specific primers containing
HindIII sites at the end (primer 711 for P1, 5
CTAAAGCTTGAATTCATTATAACTCTG 3
; primer 713 for P3, 5
CTAAAGCTTATTATTAAAAATGTTGCT 3
; primer 715 for P5, 5
CTAAAGCTTTCAAGTAGCGAAACAAAA 3
) were synthesized and used for PCR.
Amplified DNA fragments were digested with HindIII and
SalI and subcloned into the pCAT-basic vector (Promega) cut
with HindIII and SalI. To construct E-series reporters (E1, E1m, E2, and E2m), oligonucleotides were synthesized, annealed, and subcloned into pBLCAT2. Correct clones were selected by
DNA sequencing. Oligonucleotides used were as follows: for the E1
construct, primer E153 (5
AGCTTTATGTACATATGCACTACATATGCAATTATATACATATGTGAACAG 3
)
and primer E135 (5
GATCCTGTTCACATATGTATATAATTGCATATGTAGTGCATATGTACATAA 3
); and for the
E1m construct, primer E1m53 (5
AGCTTGTAGATATCCACTAGATATCCAATAACCT-3
) and primer E1m35
(5
-CTAGAGGTTATTGGATATCTAGTGGATATCTACA-3
); and for the E2
construct, primer E253 (5
GATCCTTAAAATCAAGTGTGCGAAAATCTGCACTTGAGCGCCACTTGACAACAG 3
) and primer E235 (5
GATCCTGTTGTCAAGTGGCGCTCAAGTGCAGATTTTCGCACACTTGATTTTAAG 3
); and For
the E2m construct, primer E2m53 (5
GATCCTTAAAATGAAGTCTGCGAAAATCTGGACTTCAGCGCGACTTCACAACAG 3
) and primer
E2 m35 (5
GATCCTGTTGTGAAGTCGCGCTGAAGTCCAGATTTTCGCAGACTTCATTTTAAG 3
). For the construction of the twist expression vector
pRC/CMV-Twist, pKS-Twist was digested with NotI and
ApaI, and the gel-eluted DNA fragment (1.6 kb) was subcloned
into the pRC/CMV vector (Invitrogen). To construct the truncated form
of the twist expression vector CMV-TwiH, twist
cDNA (0.57-kb EcoRI DNA fragment; amino acids 324
490)
was obtained from pGBT-TwiH3 and subcloned into the
EcoRI site of the NK-4N expression vector4 that
contains 109 bp of 5
-untranslated region of NK-4 and the initiator codon. To construct the NK-4 expression vector
pRC/CMV-NK4, specific primers were synthesized (primer 466, 5
ACGGCGGCCGCCGAGATTCCAATTCAAGT 3
; primer 465, 5
CTGGGGCCCTTAATCGTCGTCCTTGTAGTCAGCCATGTGCTGCATCTGTTGC 3
) and used for
PCR. Primer 465 has a coding sequence for the Flag peptide (IBI) so
that the expressed NK-4 protein can be tagged with the Flag peptide.
Amplified DNA fragments were digested with NotI and
ApaI, and subcloned into the corresponding sites of
pRC/CMV.
A PCR-based method was used to
generate mutations within the E1 or E2 cluster for the construction of
P1E1m, P1E2m, and P1E1mE2m (all of the three E-box sequences are
mutated). For the P1E1m construct, two separate DNA fragments (223 bp,
from 1336 to
1114; 986 bp, from
1122 to
137) were amplified by
PCR with specific primers (primer 711; primer 921, 5
TTGCTCGAGTAGTGCGTACGTACATACAGTACACA 3
; primer 923, 5
CTACTCGAGCAATTATATACGTACGTGAACACGTTTTTGGT 3
; primer 724, 5
TAGGGATCCAACCGTTAGCGCTTCCGT 3
). Amplified DNAs were digested with
HindIII-XhoI and
XhoI-BamHI, respectively, and ligated into the
pBluescript vector cut with HindIII-BamHI. Subclones were sequenced, and selected plasmids containing the mutated
E-box sequences within the E1 cluster were digested with HindIII-XbaI. DNA fragments were eluted from an
agarose gel and subcloned into the HindIII-XbaI
sites of the pCAT-basic vector. To construct P1E2m and P1E1mE2m
reporters, the pKS-P4E2m plasmid containing mutations within the E2
cluster was constructed first. To construct the pKS-P4E2m, primers were
synthesized (primer 925, 5
GGTAAGCTTGCTTAGTACACTCTTAAAATGAAGGTCTGCGAAAATCTGGACTTCAGCGCGACTTCACAACCGTTTAATACACA 3
; primer 724, 5
CAGGGATCCAACCGTTAGCGCAACCGT 3
) and used for PCR. Amplified DNA fragments were digested with HindIII and
BamHI and subcloned into a pBluescript vector. Mutations
within the E2 cluster were confirmed by DNA sequencing. From this
construct fragment C (771 bp, from
907 to
137) containing mutations
within the E2 cluster was amplified with specific primers (primer 927, 5
CCACGATTTATTTATTTGTTAGCTTAGTACACTCTTAAAATG 3
; primer 750). Two
additional DNA fragments (471 bp, from
1336 to
866) containing either the wild type (fragment A, from P1 template DNA) or the mutated
E-box sequence (fragment B, from P1E1m DNA) within the E1 cluster were
amplified from different template DNAs with specific primers (primer
711 and primer 928, 5
CATTTTAAGAGTGTACTAAGCTAACAAATAAATAAATCGTGG 3
).
Mixed DNA fragments (A and C for P1E2m; B and C for P1E1mE2m) were
denatured and re-annealed. DNAs (41 nucleotide overlap) were gap-filled
with Vent DNA polymerase (New England Biolab). Gap-filled DNAs were
subjected to PCR (30 cycles of 1 min at 95 °C, 1 min at 55 °C, 2 min at 72 °C) with primers 711 and 724 to generate full-length DNA
fragments (1.2 kb, from
1336 to
137). Amplified DNAs were
gel-eluted and subcloned into pBluescript vector. Mutated regions of
subclones were sequenced again with specific primers. Selected clones
were digested with HindIII and XbaI, and
gel-eluted DNA fragments were subcloned into the pCAT-basic vector.
Gel-shift assays were performed
with partially purified fusion proteins (155 ng) and
32P-labeled probes (5 × 104 cpm, 5-10
fmol) in binding buffer A containing 25 mM HEPES (pH 7.5),
3 mM MgCl2, 1 mM EDTA, 0.5%
Nonidet P-40, 10% glycerol, 1 µg of poly[d(I-C)]. Reactions were
incubated at room temperature for 15 min and analyzed on 4%
polyacrylamide gels in 0.25 × Tris borate buffer. To prepare
oligonucleotides probes, equimolar amount of oligonucleotides were
annealed and end-labeled with T4 kinase or Klenow fragment. To make the
F35 probe, DNA amplified by PCR with specific primers was subcloned
into the HindIII-BamHI sites of the pBLCAT2
vector. From this construct the F35 DNA fragment (78 bp from 886 to
809) was gel-eluted, dephosphorylated, and end-labeled by T4 kinase.
Footprinting assays were performed as described previously (49). The
F23 (for Twist footprinting) or F33 (for NK-4 footprinting) plasmid DNA
was digested with HindIII (in the case of minus-strand
labeling, BamHI was used), and digested DNAs were
dephosphorylated with alkaline phosphatase. Dephosphorylated DNAs were
redigested with BamHI (in the case of minus-strand labeling, HindIII was used). DNA fragments were eluted from a gel, and
the concentration of DNA was measured. End labeling was done by T4 kinase. Binding reactions were performed with fusion proteins and
labeled DNA (12.5 fmol) in 20 µl of binding buffer A. After incubation for 15 min at room temperature, CaCl2 (final 0.5 mM) and DNase I (0.05 unit) were added to the binding
reactions for DNase I digestion. Reactions were stopped after 1 min
incubation by adding stop buffer and were analyzed on an 8% denaturing
polyacrylamide gel.
CV-1 cells were grown in
minimal essential medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum, and Drosophila S2 cells were grown
in M-3 medium (Quality Biologicals Inc.) supplemented with 10% fetal
bovine serum. Cells (2 × 105 per 60-mm dish) were
transfected with plasmid DNAs (total 12 µg per dish; amount of DNA
was adjusted with salmon sperm DNA) by the calcium phosphate
precipitation method. Usually, 3 µg of reporter plasmid, 1 µg of
expression vector, and 1 µg of -galactosidase expression vector
(pCMV
; CLONTECH) were used unless indicated. For
cotransfections with two different expression vectors, the pRC/CMV
empty vector was used to adjust the total amount of expression vector.
Cells were harvested 48 h after transfection, washed, and finally
solubilized in 60 µl of lysis buffer (250 mM Tris-HCl (pH
8.0)). Cells were lysed with three cycles of freezing and thawing, and
cell extracts (supernatants) were collected after centrifugation and
used for
-galactosidase activity and CAT assays. Twenty µl of the
extracts were used to measure CAT activities with a CAT enzyme-linked
immunosorbent assay KIT (Boehringer Mannheim) according to
manufacturer's protocol. Transfection efficiencies were normalized
with
-galactosidase activity.
Standard
procedures were used for P-element-mediated germ line transformation
(50). The y w67c23 strain was used for embryo
injections with P-elements P1LacZ (wild type), P1E1mLacZ (E1 mutation),
P1E1E2mLacZ (E2 mutation), and P1E1mE2mLacZ (E1 and E2 mutations),
respectively. P-elements were constructed by inserting corresponding
NK-4 promoter and upstream regions (EcoRI and
BamHI double-digested DNA fragments) from P1, P1E1m, P1E2m,
and P1E1mE2m reporters that were used for transient expression assays
in cultured cells into CaSpeR -galactosidase vector. A minimum of
four lines was established for each construct. For the characterization
of reporter gene expressions in transgenic flies, embryos from each fly
stock were collected, dechorionated, and fixed as described previously
(51) and subjected to incubation with the anti-
-galactosidase
primary antibody (Cappel; 1:5000 dilution). Signals (brown precipitate)
were developed with peroxidase (horseradish peroxidase)-conjugated
secondary antibody (Life Technologies, Inc.) and diaminobenzidine.
Because the potential hierarchical relationship between
twist and NK-4 was suggested previously (19, 26),
the 5 upstream region of the NK-4 promoter was sequenced
and found to contain nucleotide motifs such as E-boxes. Using two
subcloned plasmids (a 1.62-kb EcoRI insert and a 2.3-kb
EcoRI-BamHI insert, respectively) which contain
the 5
upstream region, the first exon, and part of the first intron,
serial deletion mutant plasmids were generated and used as template
DNAs for DNA sequencing. A total of 3 kb of sequence from the upstream
region was obtained by aligning the overlapping sequences. The portion
of the upstream sequence that was determined is shown in Fig.
1A. We reasoned that this region might
contain functionally important regulatory sequences for the expression
of NK-4, since two deletion alleles within this region
result in lethality (30). In the proximal region (from
337 to
137)
to the initiator codon, we detected the basal promoter activity by an
analysis of transient expression of reporter constructs in
Drosophila S2 cells (data not shown). In addition to many
putative regulatory sites, we found 11 E-box (CANNTG) sequences that
are putative binding sites for the bHLH type transcription factors such
as MyoD and Twist. Of interest are two clusters of E-box sequences (see
Fig. 1A; E1 cluster, 3 copies of ACATATG from
1134 to
1101; E2 cluster, 3 copies of CACTTGA from
868 to
831). If these
two clusters of E-box sequences have a regulatory function in
NK-4 expression, then they may be conserved in other species
during evolution. To test this, we cloned the NK-4 homologue of D. virilis (DvNK-4). Interestingly, several
clones contained both the NK-4 and NK-3 homeobox
genes, suggesting that the D. virilis genome also has the
same homeobox gene cluster as D. melanogaster (28). The
deduced amino acid sequences of the DvNK-4 and
DvNK-3 homeodomains are nearly identical to those of
D. melanogaster, and the gene structure is also
similar.4 In addition, we found that the E-box clusters are
also conserved in D. virilis (Fig. 1B).
Conservation of the E-box clusters during evolution suggests that these
sequences may have an important regulatory function for NK-4
expression.
Twist Binds to the E1 Cluster, but not to the E2 Cluster DNA
Various bHLH transcription factors can bind to the E-box
sequence as homo- or heterodimers (52, 53). We observed that nuclear
extracts from the Drosophila embryos contain DNA binding proteins for both E1 and E2 cluster DNA, indicating that regulatory proteins such as bHLH transcription factors might bind to these E-box
clusters during embryogenesis (data not shown). If the homodimer of the
Twist protein is functional in DNA binding, we should be able to detect
binding activity of the Twist protein to the E-box sequence. As an
initial step toward determining whether Twist activates NK-4
directly, we first examined whether Twist could bind to the E-box
clusters located in the 5 upstream region of the NK-4
promoter. The Twist protein used in the DNA binding studies was
prepared by expressing twist cDNA in E. coli
as a GST:Twist fusion protein, which was partially purified using a
glutathione-Sepharose column. The GST:Twist fusion protein was analyzed
for its DNA binding properties by gel-shift assays using the labeled E1
or E2 DNA containing two copies of the E-box sequence within the E1 or
E2 cluster region as a probe. We found that the GST:Twist fusion
protein bound to the E1 cluster specifically, presumably as a homodimer
(Fig. 2A, lanes E1 PROBE). However, we could
not detect any binding activity to the E2 cluster DNA (Fig. 2A,
lanes E2 PROBE). We also performed DNase I footprinting assays
with a labeled F23 DNA fragment (300 bp from
1186 to
887)
containing the E1 cluster. Results shown in Fig. 2B
demonstrate that the Twist protein leads to specific footprints on the
E1 cluster that are consistent with the results of the gel-shift
experiments shown in Fig. 2A. These results demonstrate that
Twist binds to the E1 cluster DNA but not to the E2 cluster DNA.
Dependence of NK-4 Activation by Twist on the E1 Cluster
We
next asked whether the specific binding of a GST:Twist fusion protein
to the E1 cluster DNA in vitro is functionally related to
NK-4 regulation by twist in cultured cells. For
this purpose, we employed transient expression assays in cultured CV-1
cells, since previously we found that the Drosophila S2
cells contain endogenous twist and NK-4
activities. We constructed a Twist expression vector, pRC/CMV-Twist,
containing the complete twist cDNA fragment driven by a
strong CMV promoter. For the construction of the F23 and F33 reporter
plasmids containing either the E1 (F23 DNA fragment) or the E2 cluster
region (F33 DNA fragment, 300 bp from 1036 to
737), PCR-amplified
DNAs were subcloned into the pBLCAT2 vector, in which the
chloramphenicol acetyltransferase (CAT) gene is driven by the
heterologous thymidine kinase promoter. When we measured CAT activities
from cell extracts cotransfected with reporter plasmids and with the
Twist expression vector, increased CAT activities were observed only in
cell extracts cotransfected with the F23 reporter (Fig.
3A). Cotransfection with the F33 reporter
containing the E2 cluster resulted in no increase in CAT activity. To
investigate whether the E-box sequences of the E1 cluster within the
F23 construct are responsible for the activation of this reporter, we
constructed the F23E1m reporter containing a mutated sequence of the E1
cluster and measured CAT activity after cotransfection. Indeed,
mutations within the E1 cluster abolished the increase in CAT activity
that was seen in the F23 transfection following twist
expression, suggesting that the E-box sequences (CATATG) within
the E1 cluster are necessary for the activation of the reporter gene by
Twist (Fig. 3A). Similarly, we constructed reporters (E1 and
E1m) in which the E1 cluster alone (from
1139 to
1095) or the
mutated E1 cluster are attached to pBLCAT2 and tested transactivation
of these reporter genes by twist expression. The results
showed that the wild type E1 cluster sequence was sufficient to induce
an increased CAT activity when cells were cotransfected with the
twist expression vector, whereas the mutated E1m sequence
was not (Fig. 3A). These results demonstrate that the
sequence requirements for Twist binding to the E1 cluster closely
parallel those necessary for NK-4 activation by
twist.
NK-4 Is a Direct Transcriptional Target for Twist in Cultured Cells
Finally, we tested whether Twist is able to activate the
NK-4 homeobox gene directly in cultured cells. To this end, we used the
NK-4 promoter and the 5 upstream region for the reporter constructs. The wild type P1 construct was generated by inserting 1.2 kb of DNA (from
1342 to
137) from the NK-4 promoter and the 5
upstream region into the pCAT-basic vector. For the construction of the mutant P1E1m, we introduced mutations within the E1 cluster. Following cotransfection of cells, a 6-fold increase in CAT activity was observed with the P1 construct which was dependent upon the twist expression (Fig. 3B). In contrast, the
mutant reporters (P3 and P1E1m) did not show a significant increase in
CAT activities compared with that of the P1 reporter construct (Fig.
3B). In addition, transfections with a truncated form of the
twist expression vector, CMV-TwiH, that contains a bHLH
domain but lacks a transcriptional activation domain fail to show
NK-4 activation, indicating the direct involvement of the
Twist protein in transactivation of NK-4 (Fig.
3C).
The residual CAT activity (see Fig. 3B; 1.8-fold increase)
in P3 and P1E1m prompted us to search for other putative
twist regulatory sites. We tested potential Twist binding to
E-box sequences including those that might occur in the 5 upstream
region (up to 3 kb) of the NK-4 promoter, by gel-shift
assays (Fig. 4). We found that the Twist protein could
bind to the E-box sequences with differential affinities under our
experimental condition (strong binding, Fig. 4, lanes 1 and
7; moderate binding, lanes 8 and 10;
weak binding, lanes 5 and 9; no binding,
lanes 2-4, and 6). These strong binding sites
(lanes 1 and 7) also exist in the
rhomboid neuroectodermal element (47), and one binding site
with medium affinity (lane 10) was found in the
snail proximal enhancer element (54). As far as the Twist
binding site in the NK-4 promoter and the 5
upstream region
(up to 3 kb) is concerned, we did not find any strong binding sites
except for the E-boxes (CATATG) within the E1 cluster (Fig. 4,
lanes 1-5). This E-box sequence was found in two other
locations (TACATATGC from
1657 to
1649, data not shown; AGCATATGA,
from
444 to
436), and a weak binding site (AGCAGCTGG from
675 to
667) was also found. These two binding sites within the F53 DNA
fragment (from
738 to
438) may explain the residual CAT activities
seen in P1E1m and P3 transfections. Indeed, coexpression of F53
containing these two sites (300 bp, from
736 to
436) and
twist showed a mild increase in CAT activity (data not
shown). Taken together, these results strongly suggest that
NK-4 is a direct transcriptional target of twist
in cultured cells.
The E2 Cluster Is Responsible for the Autoregulation of NK-4
Because autoregulation of homeobox genes has been described
previously, we examined whether NK-4 could also autoregulate
its own gene in cultured cells. To this end, various overlapping
upstream regions (300-bp DNA fragment each) of the NK-4
promoter were amplified and subcloned into the pBLCAT2 reporter (Fig.
5, F23-F53), and the effect of
NK-4 on CAT activity was measured. We observed that CAT
activities from cell extracts cotransfected with the F33 or F43
reporters were increased in the presence of the NK-4
expression vector pRC/CMV-NK4 (Fig. 5A). Additionally, we
observed increased CAT activity in cells cotransfected with F3 and the
NK-4 expression vector. These results indicate that the
overlapping region between the two constructs (F3 DNA fragment, 150 bp
from 888 to
737) contained cis-acting DNA elements
responsible for the autoregulation by NK-4. To determine the
NK-4 binding sites within this region, we expressed a GST:NK-4 fusion
protein in E. coli and used it for DNA binding studies.
Using gel-shift assays we found strong binding activities to the F35
DNA fragment (from
886 to
809) in which the E2 cluster sequence is
located (Fig. 6A). This binding was
eliminated by competition with oligonucleotides containing two copies
of the E-box sequence within the E2 cluster region (from
852 to
827). DNase I footprinting assays also showed protection of the E2
cluster region in the presence of the NK-4 protein (Fig. 6B), suggesting that the E-box sequences within the F35
region are binding sites for the NK-4 protein. This finding was again confirmed by competition assays with oligonucleotides containing a
mutated sequence within the E-box. None of the tested sequences could
compete with the wild type E-box sequence except MUT3 (CACTTAA) under
our experimental condition (Fig. 6C). In fact, we could see
the protection of the additional E-box sequence (TCAAGTG, from
963 to
957) which has the same sequence as that in the E2 cluster (Fig.
6B). These results demonstrate that the E-box sequences in
the E2 cluster are strong binding sites for the NK-4 homeodomain.
Having demonstrated NK-4 protein binding to the E2 cluster, we sought to examine the functional relevance of this phenomenon to NK-4 autoregulation. We tested a set of reporters (F3E2m, E2, and E2m) containing the F3 region with a mutated E-box sequence within the E2 cluster (F3E2m) and containing the wild type E2 sequence (E2), or mutated E2 sequence (E2m) alone (Fig. 5). We found that mutations within the E2 cluster completely abolished the CAT activity seen in the F3 transfection (Fig. 5B). Similarly, whereas transfection with E2 resulted in increased CAT activity, transfections with E2m did not show any increase in reporter gene expression. Consistent with these results, the wild type P1 reporter showed an increased CAT activity in the presence of NK-4. In contrast, the deletion mutant (P5) and mutations within the E2 cluster (P1E2m) resulted in decreased CAT activities (Fig. 5C). These results demonstrate that the E2 cluster is responsible for NK-4 autoregulation.
Both the E1 and the E2 Cluster Are Required for NK-4 Activation in Visceral Mesodermal Cells in VivoTo address the in
vivo function of the two clusters of E-box sequences, we
established transgenic flies containing P-elements (wild type, P1LacZ;
E1 mutation, P1E1mLacZ; E2 mutation, P1E2mLacZ; E1 and E2 mutations,
P1E1mE2mLacZ). For constructions of the P-elements, DNA fragments
containing the NK-4 promoter and the 5 upstream regions
from P1, P1E1m, P1E2m, and P1E1mE2m, respectively, were subcloned into
the CaSpeR P-element vector. In these constructs, the lacZ
reporter gene is driven by the same NK-4 promoter and enhancer region that were characterized in transient expression assays
in cultured cells. Expressions of the
-galactosidase marker was
monitored with immunohistochemistry during embryogenesis. As shown in
Fig. 7, the wild type NK-4 reporter gene is
expressed in visceral mesodermal cells at late stage 11 embryos
(arrow, A and B), which is consistent
with tinman expression in those cells. These results
indicate that the enhancer region that we have characterized in
cultured cells is sufficient for NK-4 activation in visceral
mesodermal cells in vivo. When we mutated either the E1 or
E2 cluster,
-galactosidase activity was abolished in these cells
(Fig. 7, D-F), demonstrating that both the E1
and E2 clusters are responsible for the NK-4 activation in
visceral mesodermal cells. Furthermore, together with data that Twist
and NK-4 bind to these clusters in vitro, and that those
bindings result in reporter gene activations in cultured cells, these
in vivo results suggest that NK-4 is a direct
transcriptional target for Twist in visceral mesodermal cells and
support the notion that twist function is also required for
the subdivision of mesoderm during Drosophila embryogenesis.
Ectopic
-galactosidase expression in midline cells was also observed
(Fig. 7, A-C, arrowhead), and the
-galactosidase
protein, presumably because of stability of
-galactosidase, remained
at stage 13 embryos in visceral muscle cells (Fig. 7C,
arrow). Interestingly, we found that in embryos from transgenic
flies containing P-elements that have mutations in either the E1 or E2
cluster (Fig. 7, D-F) ectopic reporter gene
expression in somatic muscle cells was observed. Ectopic expression of
lacZ reporter in the P1E1mLacZ embryos was weaker than in
embryos from other transgenic lines (P1E2mLacZ). These results suggest
another function for the E-box clusters in NK-4 regulation,
that is that both enhancer elements are required for NK-4
repression in cells that do not express NK-4 normally. In addition, since in these embryos reporter gene activation in visceral mesodermal cells disappeared, both elements are functionally connected and are required for the NK-4 activation in visceral
mesodermal cells. The idea that both elements are required for the
NK-4 activation was tested in transient expression assays
(Fig. 8). Indeed, in the case of the wild type P1
construct, cotransfection of both NK-4 and twist
expression vectors showed an increase in CAT activities, whereas either
mutation in the E1 or E2 cluster (P1E1m, P1E2m, and P1E1mE2m)
eliminated CAT activation despite the presence of NK-4 and
twist expression vectors (Fig. 8). Taken together, the results suggested multiple function of the E-box clusters for the
NK-4 regulation (Fig. 9).
In the present study, we examined transcriptional control of
NK-4 and show that two distinct clusters of E-box sequences
mediate gene activation in visceral mesodermal cells. Several lines of evidence support the idea that the direct activation of NK-4
by twist also occurs during embryogenesis. First, the
temporal and spatial expression patterns of NK-4 and
twist show that both genes are expressed in the presumptive
mesoderm in cellular blastoderm stage embryos and in the mesodermal
layers after gastrulation (11, 19, 30). Moreover, the onset of
NK-4 expression follows the appearance of the Twist protein.
Also, cells that express NK-4 are included within regions
that express twist. Second, in the absence of
twist, NK-4 is not expressed (19), suggesting that Twist is responsible for its activation, either directly or
indirectly. Third, we show that ectopic expression of Twist in cultured
cells induces activation of reporter genes driven by the 5 upstream
region and the NK-4 promoter (Fig. 3). And we demonstrate
that in addition to in vitro binding of Twist protein to
E-box sequences within the E1 cluster of the 5
upstream region of the
NK-4 promoter (Fig. 2), mutations within the E1 cluster abolish activation of NK-4 by Twist (Fig. 3B).
Also, the expression of a truncated form of Twist does not activate the
reporter gene (Fig. 3C), indicating that direct binding of
Twist protein to the E1 cluster DNA is required for NK-4
activation. Finally, transgenic animal analysis showed that both the E1
and E2 clusters are required for the expression of NK-4 in
visceral mesodermal cells in which relatively low concentrations of
Twist exist during embryogenesis (Fig. 7). Taken together with the
genetic data, the results shown here establish a functional role for
Twist in the direct activation of NK-4 in visceral
mesodermal cells.
twist encodes a bHLH transcription factor (11) that binds to E-box (CANNTG) sequences (47, 54). Domain analysis of the Twist protein using GAL4:Twist chimeras in yeast also indicated that the glutamine-rich regions contained a transcriptional activation domain (55). In vivo, twist is activated by dorsal to give a graded distribution that tails off laterally at the cellular blastoderm stage (13-16). Later in the subdivision of the mesoderm, high levels of Twist expression is maintained in cells that will give rise to somatic muscles, whereas relatively lower amounts are expressed in progenitors of other derivatives (27). Therefore, Twist may regulate different target genes by differential DNA binding affinities depending on the concentration of the Twist protein. We showed that Twist can bind to target DNAs with differential DNA binding affinities (Figs. 2 and 4) and that Twist protein binds to the E1 cluster and activates NK-4 in visceral mesodermal cells (Figs. 3 and 7). Since we found that the E1 cluster element contains strong binding sites for Twist and is required for the NK-4 activation in visceral mesodermal cells, we propose that the relatively low concentration of the Twist protein may be sufficient for the recognition of the NK-4 promoter and the visceral mesoderm enhancer elements such as the E1 cluster. Therefore, our results provide, for the first time, direct evidence supporting the notion that twist function is also required for the subdivision of the mesoderm during Drosophila embryogenesis (27).
Is the concentration gradient of Twist protein sufficient for the selection of target genes during mesodermal cell specification? Heterodimerization and post-translational modification may be other important factors for Twist function, although little is known about modification of Twist protein, such as phosphorylation, and about Twist's partner for heterodimerization. So far, our data suggest that the Twist homodimer is sufficient for DNA binding (Figs. 2 and 4) and for the activation of NK-4 that was seen following the wild type twist expression (Fig. 3B). Nevertheless, our transgenic animal data showed that another E-box cluster (the E2 cluster), which does not bind Twist homodimer, is absolutely required for NK-4 activation in visceral mesodermal cells and is functionally linked with the E1 cluster (Figs. 7 and 8). Therefore, although a certain concentration of Twist protein itself is important to select target genes (27), we prefer the possibility that other transcription factors such as NK-4, at least in the case of the visceral mesodermal cell specification, may cooperate to regulate target genes, thereby specifying the subdivision of the mesoderm. Yet it remains to be determined whether the Twist homodimer may directly or indirectly interact with other transcription factors that bind to the E2 cluster or whether Twist may form heterodimers with unknown bHLH partners to find correct target genes during mesodermal cell differentiation. It was shown previously that dorsal-bHLH interactions are important for initiation of the embryonic mesoderm (54, 56, 57).
We demonstrate that the NK-4 protein binds to the E-box sequence (TCAAGTG) within the E2 cluster (Fig. 6) and autoactivates NK-4 (Fig. 5). It is worth noting that the NK-4 protein strongly binds to this target sequence rather than one containing the 5-TAAT-3 core. Recently, it was shown that similar binding sites were recognized by NK-2 class homeodomain transcription factors such as NK-2 (TNAAGTGG (58)), TTF-1 (TCAAGTGT (59), CEH-22 (CGCTAAAGTG (60)), and NKx-2.5 (TNAAGTG (61)). Interestingly, all members of the NK-2 family of homeodomains have a tyrosine residue at position 54 (46, 62, 63), suggesting that this residue may have an important function in recognizing target DNA sequences (64). Positive autoregulation is seen in many homeobox genes (65-70), and tissue-specific negative autoregulation is also seen in Ubx (71). Likewise, we demonstrate that NK-4 up-regulates its own gene by binding to the E2 cluster. The NK-4 protein has both activator and repressor domains,4 suggesting that NK-4 can act as either a transcriptional activator or repressor molecule. Indeed, NK-4 can down-regulate a specific reporter gene (Fig. 5C; P1E2m), suggesting that, depending on chromatin context, it can act as a transcriptional repressor. Yet it remains to be seen whether NK-4 down-regulates its own gene or other unknown target genes in a tissue-specific manner.
As discussed above, one function of the E2 cluster is to serve as an enhancer element for NK-4 activation in visceral mesodermal cells, which is shown by the transgenic animal data (Fig. 7). Because the E2 cluster that is recognized by NK-4 also contains E-box sequences, it is conceivable that in cells that do not express NK-4, this cluster may serve as a negative regulatory element for unknown bHLH proteins. We have shown that mutation in either the E1 or E2 cluster abolished the expression of the reporter gene both in visceral mesodermal cells and in cultured cells indicating that two distinct clusters of the E-box elements are indispensable for NK-4 activation and are functionally connected. Furthermore, in embryos carrying mutant reporters, ectopic expression was observed in a subset of somatic muscle cells (Fig. 7, D-F). These results provide a third function for the E-box clusters, that is they are actively involved in NK-4 repression in cells that do not normally express NK-4 (Fig. 9). It is of note that the NK-3 protein acts as a transcriptional repressor and is also able to bind to the same DNA sequences as NK-4.4 Therefore, it is probable that the E2 cluster is responsible for NK-4 repression by other transcription factors such as NK-3 in visceral mesodermal cells.
Dorsoventral axis formation in the Drosophila embryo is controlled by a cascade of transcription factors (1, 2). Downstream target genes may use separable, but sometimes tightly linked, cis-acting regulatory elements in combination with a homologous core promoter to be expressed in a temporally and spatially regulated manner (Figs. 8 and 9; Refs. 72 and 73). Perhaps gene interactions among mesodermal genes, once activated by upstream genes such as twist, are important for these tight regulations during mesodermal cell specification (35). The recent finding that twist function is also required for the subdivision of the mesoderm (27) strengthens the importance of our results which provide the first evidence that twist function is directly required for target gene activation in visceral mesodermal cells. Inductive signals such as Dpp and Wingless from other germ layers also have pivotal roles for the subdivision of the mesoderm (3, 5, 7-9, 74). Since the two clusters of E-box sequences that we have characterized here only explain NK-4 activation in visceral mesodermal cells, other regulatory elements that are involved in the activation of NK-4 in other tissues such as the dorsal vessel remain to be characterized. Further characterizations of NK-4 regulation should offer insights into the complex mechanisms controlling mesodermal cell specification.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF00436.
We are grateful to Drs. Robert Adelstein and Marshall Nirenberg for their support, discussions, and encouragement during this study. We also thank Dr. Mark Ptashne for providing GAL4 vectors for domain analysis. We gratefully acknowledge our colleagues in the Kim lab and members of the Adelstein lab for help and discussion.