J Biol Chem, Vol. 274, Issue 42, 29655-29665, October 15, 1999
Regulation of Metallothionein Gene Transcription
IDENTIFICATION OF UPSTREAM REGULATORY ELEMENTS AND TRANSCRIPTION
FACTORS RESPONSIBLE FOR CELL-SPECIFIC EXPRESSION OF THE
METALLOTHIONEIN GENES FROM CAENORHABDITIS ELEGANS*
Lori H.
Moilanen
§,
Tetsunari
Fukushige¶, and
Jonathan H.
Freedman
**
From the Nicholas School of the Environment, Duke
University, Durham, North Carolina 27708, the ¶ Department
of Biochemistry and Molecular Biology, University of Calgary,
Calgary, Alberta T2N 4N1, Canada, and the
Department of Microbiology, Duke University Medical
Center, Durham, North Carolina 27710
 |
ABSTRACT |
Metallothioneins are small, cysteine-rich
proteins that function in metal detoxification and homeostasis.
Metallothionein transcription is controlled by cell-specific factors,
as well as developmentally modulated and metal-responsive pathways. By using the nematode Caenorhabditis elegans as a model
system, the mechanism that controls cell-specific metallothionein
transcription in vivo was investigated. The inducible
expression of the C. elegans metallothionein genes,
mtl-1 and mtl-2, occurs exclusively in intestinal cells. Sequence comparisons of these genes with other C. elegans intestinal cell-specific genes identified
multiple repeats of GATA transcription factor-binding sites
(i.e. GATA elements). In vivo deletion and
site-directed mutation analyses confirm that one GATA element in
mtl-1 and two in mtl-2 are required for
transcription. Electrophoretic mobility shift assays show that the
C. elegans GATA transcription factor ELT-2 specifically binds to these elements. Ectopic expression of ELT-2 in non-intestinal cells of C. elegans activates mtl-2
transcription in these cells. Likewise, mtl-2 is not
expressed in nematodes in which elt-2 has been disrupted.
These results indicate that cell-specific transcription of the C. elegans metallothionein genes is regulated by the binding of
ELT-2 to GATA elements in these promoters. Furthermore, a model is
proposed where ELT-2 constitutively activates metallothionein expression; however, a second metal-responsive factor prevents transcription in the absence of metals.
| |
INTRODUCTION |
Metallothioneins (MT)1
are a family of structurally related, low molecular weight,
cysteine-rich proteins (1). The precise physiological role of MT has
not been elucidated. However, evolutionary conservation across many
phyla suggests that it serves important roles in cell function.
Proposed roles include the following: (a) participation in
maintaining the homeostasis of essential trace metals; (b)
sequestration of toxic metals, such as cadmium and mercury;
(c) acting as a reservoir of essential metals that can be
donated to other metalloproteins; and (d) protection against intracellular oxidative damage (2).
Exposure of cultured cells or whole organisms to transition metals,
ionizing radiation, heat-shock, or oxidative stress induces MT
transcription (1, 3-5). Metallothioneins typically occur in multigene
families. The mammalian MT family consists of four members designated
MT-I to MT-IV (6). It has been commented that all organisms express MT
or MT-like proteins. However, not all tissues within an organism will
express all MT isoforms. Numerous studies indicate that individual MT
family members display specific cellular patterns of expression.
Typically, MT-I and MT-II genes are coordinately expressed. It has been
shown, however, that MT-2A mRNA levels do not increase in response
to cadmium exposure in human proximal tubule cells (7). Expression of
the human MT-IB, MT-IE, MT-IF, and
MT-IG genes varies in a cell line-specific manner in
response to transition metals (8-12). Mouse MT-I is weakly expressed
in testes (13). In addition, MT-III is expressed primarily in neurons,
although its mRNA is detected in testes and prostate (14-16).
MT-IV is exclusively expressed in differentiating stratified squamous
epithelia (17).
Metallothioneins from invertebrates also show highly restricted
patterns of constitutive or metal-inducible expression. For example,
sea urchin spMTA and spMTB gene expression is
limited to aboral ectoderm and embryonic gut and oral ectoderm,
respectively (18, 19). The Drosophila Mtn and Mto
genes are primarily expressed in the midgut (20, 21). In addition,
inducible transcription of the two MT genes of the nematode
Caenorhabditis elegans occurs exclusively in the intestinal
cells of larval and adult nematodes (22).
Upstream regulatory elements and transcription factors that control
metal-inducible MT transcription have been identified in a variety of
species (2, 23). Several models have been proposed describing the
regulation of metal-inducible MT expression (24-26). However, the
mechanism(s) that regulate cell-specific MT expression have not been
extensively examined. Cell-specific expression has been explored in the
context of DNA methylation, by using hypomethylating agents such as
5-azacytidine. Correlation between hypomethylation and the stimulation
of MT gene expression has been observed for rainbow trout, mouse, and
human MT genes (8, 11, 27). Differences in the levels of
cadmium-inducible transcription between human MT-IG and
MT-IF genes in liver-derived cell lines are correlated with
single nucleotide changes in the TATA boxes in each promoter (28).
Suppression of mouse MT-III gene expression in organs other than the
brain has been attributed to CTG triplet repeats within the promoter.
However, the binding of a nuclear protein to the CTG sequence has not
been demonstrated (29).
The participation of specific transcription factors has been proposed
as a potential mechanism for the regulation of cell-specific MT
transcription. A protein that binds in a metal-dependent
manner to CCAAT-homologous sequences in the mouse MT-I promoter has
been isolated from rat liver (30). The amino acid sequence of this protein is homologous to members of the liver-enriched C/EBP protein family. However, the function of this protein has not been explored in
the context of cell- or tissue-specific regulation of MT transcription. We now report on the identification of UREs and a transcription factor
that binds to these elements, which control intestinal cell-specific
transcription of C. elegans MT genes.
C. elegans provides a powerful system for investigating the
molecular aspects of cell-specific MT transcription. The developmental and cellular biology of C. elegans is thoroughly understood
in exceptional detail (31-33). The adult C. elegans
hermaphrodite contains 959 somatic cells that comprise reproductive,
muscular, nervous, and digestive systems (31-33). The timing of cell
division and differentiation is invariant, and the developmental
lineage of each cell can be traced back to the fertilized oocyte (31). High levels of evolutionary conservation between C. elegans
and other organisms are observed in many signal transduction, gene regulatory, and developmental pathways (34-37). In addition, stable lines of transgenic nematodes that express reporter transgenes in
cell-specific and temporal patterns that mimic endogenous genes can
easily be generated (38, 39).
Two MT genes, designated mtl-1 and mtl-2, have
been identified and characterized in C. elegans (22).
Induction of mtl-1 and mtl-2 transcription,
following metal exposure, occurs exclusively in all intestinal cells at
post-embryonic stages of development. Furthermore, significant levels
of intestinal cell transcription are not observed in the absence of
stress (22). These observations suggest that both tissue-specific and
metalloregulatory factors control C. elegans MT expression.
Several non-metal inducible C. elegans genes are also
expressed exclusively in intestinal cells. These include the six
vitellogenin genes, vit-1 to vit-6, the cysteine
protease cpr-1, and the gut esterase ges-1
(40-42). Multiple copies of heptameric elements, which have the
consensus sequence CTGATAA, are present in the promoters of each of
these genes, as well as the two C. elegans MT genes (22, 41,
43, 44). Originally identified in the upstream regulatory regions of
the C. elegans vitellogenin genes, these elements are
believed to be responsible for controlling the transcription of
intestinal cell-specific genes (44, 45). Deletion or mutation of the
heptameric elements, in reporter transgenes containing vit-2,
ges-1, or cpr-1 promoters, causes either a loss of
intestinal cell reporter gene expression or expression in
non-intestinal cells (41, 43, 44, 46).
The nucleotide sequence of the heptameric element is identical to the
consensus-binding site for the GATA family of transcription factors
(GATA factors). GATA factors constitute a family of structurally related transcription factors that interact with the (A/T)GATA(A/G) consensus sequence. GATA factors are expressed in distinct
developmental and tissue-specific patterns, and their involvement in
the regulation of cell-specific gene transcription is well established
(reviewed in Simon (47) and Evans (48)).
Several GATA factors have been isolated from C. elegans:
ELT-1, ELT-2, ELT-3, and END-1 (49-52). The expression of ELT-2 is restricted to intestinal cells in embryonic and post-embryonic stages
of C. elegans development. Immunofluorescence, mutagenesis, and ectopic expression experiments suggest that it is a regulator of
intestinal cell-specific gene transcription. Furthermore, the intestine
is not properly formed in C. elegans in which the
elt-2 gene has been disrupted (45). The cell-specific
pattern of post-embryonic ELT-2 expression is identical to those of
both C. elegans MT genes. Thus, ELT-2 is a potential
regulator of mtl-1 and mtl-2 transcription.
Analysis of the 5'-regulatory regions of the C. elegans MT
genes reveals the presence of two GATA-like elements in the
mtl-1 promoter and five in mtl-2 (Table I). In
this report, the contribution of each of these elements in the
regulation of C. elegans MT transcription is examined. In
addition, the ability of the ELT-2 transcription factor to bind to the
GATA elements in the C. elegans MT promoters (a)
and effect MT transcription in vivo (b) is
investigated. GATA elements and ELT-2 are required for mtl-1
and mtl-2 transcription. In addition, ectopic expression
experiments indicate that ETL-2 alone, in the absence of added metal or
stress, is sufficient to induce C. elegans MT transcription.
Here we report on the roles of GATA elements and the associated
transcription factor in controlling cell-specific MT transcription.
Data suggest that other factors (i.e. metal sensor)
contribute to the metal-inducible expression of the C. elegans MT gene. It has not previously been reported that GATA
elements or transcription factors regulate MT transcription. Thus, the
results presented in this report provide novel insights into the
complex mechanisms that govern the expression of MT genes.
| |
EXPERIMENTAL PROCEDURES |
Growth and Culture of C. elegans--
C. elegans were
routinely maintained at 20 °C on NGM agar (1.7% agar, 25 mM potassium phosphate; pH 6.0, 50 mM NaCl, 2.5 µg/ml peptone, 5 µg/ml cholesterol, 1 mM
MgCl2, 1 mM CaCl2) plates seeded with Escherichia coli strain OP50 as a food source (53).
C. elegans used for inoculation of liquid cultures were
grown on 100-mm NGM plates and then removed by washing with M9 buffer
(22 mM KH2PO4, 42 mM
Na2HPO4, 85 mM NaCl, 1 mM MgSO4) (54). Nematodes were then added to
complete S medium (0.1 M NaCl, 50 mM potassium phosphate, pH 6.0, 5 µg/ml cholesterol, 10 mM potassium
citrate, 3 mM CaCl2, 3 mM
MgSO4, 50 µM EDTA, 25 µM
MnCl2, 10 µM FeSO4, 10 µM MnCl2, 10 µM
ZnSO4, 1 µM CuSO4), which
contained E. coli, and grown at 20 °C with constant
agitation (53).
Deletion Analysis--
Deletion analysis was used to determine
the minimal DNA sequence 5' of the mtl-1 and
mtl-2 transcription start sites necessary to confer
cadmium-inducible, cell-specific, and developmentally regulated
transcription in vivo. This analysis was accomplished by
creating a series of reporter transgenes in which successively larger
regions of the mtl-1 and mtl-2 promoters were
deleted. The reporter transgenes consisted of MT promoter fragments
that were inserted into C. elegans expression vectors. These
vectors express 
-galactosidase (lacZ) that is fused to
the nuclear targeting sequence of the SV40 large T antigen. The
mtl reporter transgenes were subsequently used to generate
transgenic C. elegans (22, 38, 55). Levels and cell-specific
patterns of cadmium-inducible -galactosidase expression were
compared between transgenic nematodes that contain reporter transgenes
regulated by promoter fragments of varying lengths.
To prepare mtl-1/lacZ reporter transgenes,
promoter fragments were excised from the
mtl-1/lacZ fusion construct pMT1.1 (22). pMT1.1
contains an ~1.7-kbp of mtl-1 that is immediately upstream of the initiator ATG. Digestion of pMT1.1 with SacII and
PmlI released an ~1.3-kbp DNA fragment from the 5'-end of
the upstream regulatory region. The 3'-overhang generated following the
SacII digestion was removed following incubation with T4 DNA
polymerase (56). After the blunt ends were joined, a reporter transgene regulated by 366 bp of mtl-1 promoter DNA was produced. This
construct was designated p366mtl-1/lacZ. A second
construct was prepared by incubating pMT1.1 with SacII and
BsaAI, removing the 3'-overhang, and then ligating the blunt
ends. This generated a 5'-deletion that removed an additional 46 bp to
yield p320mtl-1/lacZ. A third transgene was
prepared that contained 253 bp of the mtl-1 promoter, p253mtl-1/lacZ. This construct was produced by
first incubating pMT1.1 with XcaI and then isolating a
3289-bp DNA fragment, which contained 253 bp of the mtl-1
promoter attached to an ~3-kbp fragment of the lacZ
reporter gene. This fragment was then joined to a 4188-bp DNA fragment
that included the pUC19 backbone vector, the 3' unc-54
region, and the remainder of the lacZ gene (55). The 4188-bp
fragment was isolated following the sequential digestion of pMT1.1 with
SacII and treating with T4 DNA polymerase to generate blunt
ends and then digesting with EcoRV and XcaI.
A reporter transgene consisting of a mtl-2 promoter deletion
was prepared by incubating the mtl-2/lacZ fusion
construct pMT2.1 with PstI. pMT2.1 contains ~2.3 kbp of
the DNA adjacent to the 5'-end of mtl-2 attached to a
lacZ reporter gene (22). The PstI digestion
released an ~2-kbp fragment from the 5'-end of the upstream regulatory region. Following ligation of the remaining plasmid, the
resulting expression vector contained 324 bp of the mtl-2 promoter region upstream from lacZ. This reporter transgene
was designated p324mtl-2/lacZ. Next, a series of promoter
deletions were prepared by removing short regions of DNA from the
5'-end of the upstream regulatory region in p324mtl-2/lacZ
by exonuclease III digestion (56). In these reporter transgenes,
designated p292mtl-2/lacZ, p269mtl-2/lacZ,
p191mtl-2/lacZ, p183mtl-2/lacZ, p174mtl-2/lacZ, and p160mtl-2/lacZ,
-galactosidase expression is regulated by 292, 269, 191, 183, 174, and 160 bp of the mtl-2 promoter, respectively.
C. elegans were transformed with the reporter transgenes
that contained promoter deletions by microinjecting young adult N2 nematodes with recombinant plasmid DNA and the plasmid pRF4, which encodes the dominant selectable marker
rol-6(su1006), as described previously (22, 38,
57). Transgenic C. elegans were selected and the reporter
transgenes maintained as extrachromosomal arrays, as described
previously (22).
Mutational Analysis of GATA Elements--
To assess the
contribution of each GATA element in the regulation of mtl-1
and mtl-2 transcription in vivo, individual GATA elements were modified. Promoter fragments that contained modified GATA
elements were produced by oligonucleotide-directed mutagenesis (56,
58). To produce uracil-containing, single-stranded DNA templates,
genomic DNA fragments of mtl-1 and mtl-2 were
first cloned into pGEM plasmids (Promega). A 1566-bp mtl-1
fragment was isolated from pMT1.1 following a BamHI
digestion and inserted into pGEM3zf(+), which was cut with the same
restriction enzyme. This construct, designated pG3mtl1, contains 1529 bp of the region in mtl-1 that is upstream from the
transcription start site. An ~1.2-kbp mtl-2 DNA fragment
was isolated following the digestion of a genomic mtl-2
clone (22) with PstI and BstXI and then cloned into pGEM5zf(+), which was cut with the same enzymes. This construct, designated p
mtl2, includes 324 bp of the 5'-regulatory region. The
remainder of the insert consists of the coding region of
mtl-2 (22). E. coli strain CJ236
(dut
ung) was transformed with
the pGEM plasmids and uracil-containing, single-stranded DNA templates
produced by infecting the bacteria with M13K07 helper phage (56).
To generate site-directed mutations in the individual GATA elements,
antisense oligonucleotides containing 6-10-bp mismatches, which also
encoded unique endonuclease restriction sites, were used to prime
complementary strand DNA synthesis. These oligonucleotides contained
8-11 correct nucleotides at the 5'-end of the mismatch nucleotides and
13-18 correct nucleotides at the 3'-end of the mutant sequence (Tables
II and III). The sequences of the GATA elements were changed to those
previously reported to inhibit GATA-mediated transcription of
vit-2 (44). Following DNA synthesis, E. coli
DH5
(dut+ ung+) were transfected
and the double-stranded pGEM plasmids that contained the modified
promoters were recovered (56, 58). Mutations were confirmed by
restriction endonuclease digestion and nucleotide sequencing.
Mutated mtl-1 promoter fragments were excised from pG3mtl1
vectors following incubation with PmlI and
HindIII. A 432-bp mtl-1 DNA fragment, which
includes 366 bp of the mtl-1 promoter, was inserted into the
C. elegans expression vector pPD16.51 (55). To generate
mtl-2-containing lacZ expression vectors,
pmtl2 was first cut with BstXI and NcoI and an
~0.9-kbp fragment isolated. This fragment was sequentially cut with
HpaII, treated with Klenow DNA polymerase, which filled-in
the 5'-overhang, and then cut with PstI. The resulting
450-bp PstI/blunt mtl-2 fragment contains 324 bp
of the mtl-2 promoter and 150 bp of the structural region of
the gene. The structural region includes the 5'-untranslated region,
the first exon, the intron, and 33 bp of the second exon. This DNA
fragment was inserted into pPD16.51 that was cut with PstI
and SmaI. This construct expressed a fusion protein
consisting of the N-terminal 16 amino acids of MTL-2 and a 7-amino acid
linker fused to nuclear targeted -galactosidase. mtl-1
and mtl-2 control expression vectors, p1Control and
p2Control, respectively, were prepared by excising non-mutated promoter
fragments from the corresponding pGEM vectors using the cloning schemes
that are outlined above. C. elegans were transformed by
microinjecting young adult N2 nematodes with recombinant plasmid DNA
and the rol-6 selectable marker, as described above.
Ectopic Expression of C. elegans GATA-binding Transcription
Factors--
To examine the effects of ELT-2 expression on MT gene
transcription, a line of transgenic C. elegans was generated
that contained two independent transgenes,
hsp-16/elt-2 and
mtl-2/lacZ. In addition, a second line of
transgenic nematodes was developed that contained both the
hsp-16/elt-1 and mtl-2/lacZ
transgenes. In these lines of transgenic C. elegans,
heat-shock will induce the ectopic expression of the GATA factors in
most of the somatic cells (45, 59). If either ELT-1 or ELT-2 can
activate MT transcription in vivo, then heat-shock will
induce -galactosidase expression in non-intestinal cells and
embryos. Similar methods were used to study the ability of these GATA
factors to control ges-1 transcription in
Drosophila and mouse (45, 60, 61).
Transgenic C. elegans containing the
mtl-2/lacZ transgene were prepared by first
injecting unc-119(ed4) mutant nematodes with p324mtl-2/lacZ and the selectable marker pDPMM016D. The
plasmid pDPMM016D encodes the wild type form of unc-119. C. elegans that contain this transgene will not exhibit the
unc-119 phenotype (38, 62). The transgene was integrated
into the C. elegans genome following 
-irradiation and
integrated lines were outcrossed several times, as described previously
(43). The strain of C. elegans containing the integrated
mtl-2/lacZ transgene was designated JF4(mtl-2/lacZ). Lines of transgenic C. elegans containing either the hsp-16/elt-1
or hsp-16/elt-2 expression vector integrated into
the genome, designated JM53(hsp-16/elt-1) or
JM57(hsp-16/elt-2), respectively, were prepared
as described previously (45).
C. elegans containing pairs of transgenes were created by
standard genetic crosses. Strains
JM53(hsp-16/elt-1) and
JF4(mtl-2/lacZ) were crossed to produce a line of
transgenic C. elegans that contains both
hsp-16/elt-1 and
mtl-2/lacZ. This line was designated
JF6(hsp-16/elt-1, mtl-2/lacZ). Line
JF5(hsp-16/elt-2, mtl-2/lacZ), which
contains both the hsp-16/elt-2 and
mtl-2/lacZ transgenes, was generated by crossing
JF4(mtl-2/lacZ) with
JM57(hsp-16/elt-2).
Histochemical Assay for Reporter Gene
Transcription--
Transgenic C. elegans were grown on a
100-mm NGM plate seeded with E. coli for ~4 days at
20 °C. Nematodes were harvested, suspended in 10 ml of S basal
medium (54), and then incubated for 8-14 h at 20 °C with constant
agitation. For cadmium treatment, CdCl2 was added to a
final concentration of 100 µM. Following the incubation,
C. elegans were collected by centrifugation for 5 min at
1,000 × g, washed twice with M9 buffer, frozen in
liquid nitrogen, and then lyophilized.
To induce ELT-1 and ELT-2 expression prior to assaying for
-galactosidase activity, transgenic C. elegans were
heat-shocked by incubating mixed stage nematodes for 1-3 h at
34 °C. The nematodes were allowed to recover by incubating overnight
at 20 °C and then collected and lyophilized as described above.
Embryos at the 2-4-cell stage were collected from
JF5(hsp-16/elt-2, mtl-2/lacZ)
nematodes and allowed to develop for 6 h at 20 °C. They were
then heat-shocked at 34 °C for 30-min and allowed to recover
overnight at 20 °C prior to staining.
Lyophilized nematodes and embryos were fixed with acetone and stained
for -galactosidase activity using
5-bromo-4-chloro-3-indolyl--galactoside as a chromogenic substrate
as described previously (22, 63). C. elegans were incubated
in staining solutions for 1-2 h, following cadmium treatment, or ~12
h following heat-shock.
Preparation of C. elegans Extracts--
Nematode extracts were
prepared using a modification of the procedure of Land et
al. (64). Mixed stage populations of N2 C. elegans were
grown in liquid culture. Cadmium-treated nematodes were grown in 100 µM CdCl2 for 12 h prior to harvest.
C. elegans were harvested, removed by sucrose flotation, and
washed as described previously (22). Washed nematodes were suspended in
an equal volume of distilled, deionized water and immediately frozen in liquid nitrogen. Approximately 5 g of frozen C. elegans
was pulverized in a liquid nitrogen-cooled mortar. Powdered nematodes
were suspended in 5 volumes of 20 mM HEPES buffer, pH 7.6, containing 0.5 mM dithiothreitol, 4 µg/ml leupeptin, 4 µg/ml aprotinin, 4 µg/ml pepstatin, 10 mM benzamidine,
and 28 µM E-64 immediately before sonication. The
suspension was sonicated on ice for 4 min with an ultrasonic
disintegrator set at 80% duty cycle using 1-s pulses. Homogenates were
centrifuged at 100,000 × g for 1 h at 4 °C.
The supernatant fraction was collected and then filtered through
Miracloth (Calbiochem). Aliquots were immediately frozen in liquid
nitrogen and stored at 
80 °C. Protein concentrations were
determined using the Coomassie Protein Assay Reagent (Pierce).
Electrophoretic Mobility Shift Assay--
EMSAs were performed
to examine the interaction between GATA elements in the C. elegans MT promoters and C. elegans extract proteins
(a) and the GATA-binding transcription factor ELT-2
(b). Double-stranded oligonucleotide probes were generated
by first annealing complementary pairs of single-stranded
oligonucleotides. The sequences of the oligonucleotides are identical
to regions that included the GATA elements 1.1, 2.1, and 2.4 (Tables I
III). Two to four additional guanidine residues were added to the
5'-end of one of the oligonucleotide strands. The double-stranded
oligonucleotides were end-labeled by filling-in the 5'-overhangs with
[-32P]dCMP. For filling-in reactions, 10-20 pmol of
annealed oligonucleotide were combined with 33 µM each of
dATP, dGTP, dTTP; 50 µCi of [-32P]dCTP; and 20 units
of Klenow fragment in reaction buffer (10 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 7.5 mM
dithiothreitol) and then incubated for 15 min at room temperature.
Reactions were terminated by the addition of 10 mM EDTA
(final concentration) and incubation at 75 °C for 10 min.
Unincorporated nucleotides were separated from the labeled products by
Sephadex G-25 spin column chromatography.
DNA-protein binding reactions using C. elegans total protein
extracts were performed by incubating 5-10 µg of extract protein with 25-50 fmol of labeled oligonucleotide (3 × 104
cpm), 1-5 µg of poly(dI-dC), and unlabeled competitor
oligonucleotide in extract assay buffer (10 mM HEPES, pH
7.6, containing 10% glycerol, 105 mM NaCl, 2 mM MgCl2). Protein and competitor
oligonucleotides were incubated for 15 min on ice, prior to the
addition of labeled probe. After probe addition, reactions were
incubated for 30 min at 25 °C and then placed in ice prior to electrophoresis.
DNA-protein binding reactions were also performed using in
vitro transcribed and translated ELT-2 protein. ELT-2 protein was synthesized from the full-length elt-2 cDNA, contained
in pBluescript SK (50), using the TNT-coupled
transcription-translation system (Promega). Binding reactions were
performed as described previously (50). Briefly, 1 µl of the in
vitro transcription/translation reaction mixture was combined with
~50 fmol of labeled oligonucleotide (2.5-5 × 104
cpm), 0.38-0.5 µg of poly(dI-dC), and unlabeled competitor
oligonucleotide in modified Zhang Buffer (25 mM HEPES, pH
7.6, containing 10% glycerol, 50 mM KCl, 10 µM ZnCl2) (65). Incubations were performed as
described above.
Products were resolved by polyacrylamide gel electrophoresis using 6%
polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) made in 0.5× TBE
(1× TBE contains 89 mM Tris, 89 mM boric acid, 2.5 mM EDTA) that had been pre-electrophoresed for 1 h
at 150 mV. Electrophoresis of the reaction mixtures was performed at 30 mA at 4 °C for 1-4 h. Gels were then dried, and the amount of
DNA-protein complex that formed was determined by PhosphorImager analysis, using ImageQuant Software version 3.3 (Molecular Dynamics).
| |
RESULTS |
Deletion Analysis of the mtl-1 and mtl-2 Promoters--
Deletion
analysis was used as the initial step in identifying cis
regulatory elements that control the intestinal cell-specific transcription of the C. elegans MT genes. In addition, it
served to delimit the 5'-boundaries of the minimal promoters for
mtl-1 and mtl-2. Minimal promoters are defined as
the shortest region of DNA, upstream from the transcription start site,
that can regulate cadmium-inducible, intestinal cell-specific, and
developmentally modulated gene transcription.
Larval and adult C. elegans containing the
p366mtl-1/lacZ transgene constitutively expressed
-galactosidase in three pharyngeal cells. This pattern of
constitutive expression is similar to that previously observed (22).
Larval C. elegans exhibited high levels of cadmium-inducible
-galactosidase expression, whereas expression was attenuated in
adult nematodes. The intestinal cell-specific pattern and levels of
inducible reporter gene expression were identical to those previously
observed in C. elegans that contain the pMT1.1 reporter
transgene (Table IV) (22).
When p320mtl-1/lacZ nematodes were treated with cadmium,
-galactosidase expression was induced in the intestinal cells of L1
and L2 larvae. However, mtl-1 promoter activity in L3, L4, and adult intestinal cells was not observed. The results suggest that
the region between 366 and 320 bp contains a regulatory element(s)
that controls mtl-1 transcription in later developmental stages. Transgenic nematodes containing
p253mtl-1/lacZ constitutively expressed
lacZ in three pharyngeal cells; however, cadmium exposure did not induce intestinal cell transcription of the reporter transgene. The region between 320 and 253 includes one GATA element, GATA1.1 (Table I), and this result suggests that
this element may be involved in the regulation of mtl-1
transcription.
Transgenic nematodes containing the mtl-2 reporter
transgene, p324mtl-2/lacZ, were exposed to cadmium or heat
shock. The pattern of inducible mtl-2 promoter activity was
identical to that previously observed in transgenic nematodes carrying
the pMT2.1 transgene (22). -Galactosidase activity was detected
exclusively in the larval and adult intestinal cell nuclei.
In p292mtl-2/lacZ, an additional 32 bp of DNA was
removed from the 5'-end of the upstream regulatory region, which
contains sequences that are homologous to cAMP response and AP-1 UREs
(22). Treatment of transgenic C. elegans carrying
p292mtl-2/lacZ with cadmium induced reporter gene
expression in intestinal cells of larvae and adults. However, the level
of promoter activity appeared to be reduced by ~50% compared with
that of transgenic nematodes containing either pMT2.1 or
p324mtl-2/lacZ. This result suggests that these
potential regulatory elements may participate in the transcriptional
control of mtl-2.
Cadmium-treated and non-treated transgenic C. elegans
containing p269mtl-2/lacZ did not express
-galactosidase in any cells. This transgene is missing an additional
23 bp from the 5'-end of the upstream regulatory region of
mtl-2, which includes the GATA2.1 element (Table I). This
result suggests that GATA2.1 may be essential for mtl-2 transcription.
Additional 5'-deletions of the mtl-2 promoter were also
examined (Table IV). -Galactosidase activity was not detected in any
cells of transgenic nematodes, in either the presence or absence of cadmium.
Deletion analysis demonstrated that all information necessary to
control cadmium-inducible, intestinal cell-specific, and developmentally regulated mtl-1 and mtl-2
transcription is present within 366 and 324 bp upstream of the
transcription start sites, respectively. These fragments were defined
as the minimal promoters. In addition, the results suggest that GATA
elements are involved in the regulation of C. elegans MT
gene transcription.
Site-direction Mutation Analysis of mtl-1 and mtl-2 GATA
Elements--
The functional contribution of GATA elements in
regulating transcription of the C. elegans MT genes was
determined by site-directed mutation analysis. The effect of mutating
each GATA element on C. elegans MT promoter activity was
assessed in vivo using transgenic nematodes.
C. elegans containing a reporter gene in which the GATA1.1
element (290 to 284) was modified did not express significant amounts of -galactosidase following cadmium exposure, relative to
levels observed in transgenic nematodes containing the p1 control transgene (Fig. 1). In contrast, mutation
of the GATA1.2 element (71 to 65) had no detectable effect on
either the level or pattern of -galactosidase expression in
cadmium-treated transgenic C. elegans. These results suggest
that the GATA1.1 element is essential for mtl-1
transcription.
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Fig. 1.
Mutational analysis of GATA elements in the
C. elegans metallothionein promoters. Potential
GATA factor binding-sites in the mtl-1 and mtl-2
minimal promoters were modified by site-directed mutagenesis, as
described under "Experimental Procedures." The specific GATA
sequence that has been modified in each transgene is indicated by ×.
Transgenic nematodes were exposed to 100 µM
CdCl2 for 8-12 h (100 µM Cd) and
then stained for -galactosidase activity. Identical populations of
C. elegans were not exposed to cadmium (0 µM
Cd) prior to staining. -Galactosidase activity in
C. elegans that contain reporter transgenes incorporating
mutated promoter sequences is expressed relative to the activity
observed in nematodes that contain unmodified reporter transgenes
(WT). Transcription start sites are indicated by
arrows. Data represent evaluation of three to five
independent experiments for each mutation.
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Cadmium treatment of C. elegans carrying transgenes
consisting of mtl-2 minimal promoters in which the sequence
of either the GATA2.1 (278 to 272) or GATA2.4 (65 to 59)
element was modified did not activate gene expression (i.e.
-galactosidase was not detected) (Fig. 1). The levels of
cadmium-inducible -galactosidase expression in C. elegans
that contained reporter transgenes in which the sequence of the
remaining three GATA elements, 2.2, 2.3 or 2.5, were changed were
identical to that of transgenic p2 control C. elegans. This
suggests that only GATA2.1 and GATA2.4 are involved in the regulation
of mtl-2 transcription. In addition, both of these elements
must be present in order for mtl-2 transcription to occur.
None of the mtl-1 or mtl-2 GATA mutations caused
either an increase in -galactosidase expression in the absence of
metal or expression in non-intestinal cells, suggesting that these UREs participate in activation rather than repression of transcription.
Binding of C. elegans Extract Proteins to GATA Elements in mtl-1
and mtl-2--
In vivo site-directed mutagenesis analysis
identified several GATA elements that are required for
cadmium-inducible transcription of mtl-1 and
mtl-2. EMSA were performed to determine if C. elegans proteins specifically bind to these sequences. A
sequence-specific DNA-protein complex was formed when a
32P-labeled, double-stranded oligonucleotide probe, which
included the GATA1.1 sequence (Tables II
and III), was incubated with C. elegans protein extracts (Fig. 2).
Formation of this complex was successfully competed by the addition of
a 50-fold molar excess of unlabeled GATA1.1 oligonucleotide (Fig. 2,
lane 3). The addition of a 50-fold molar excess of an
unlabeled oligonucleotide in which the sequence of the GATA1.1 element
was changed from CTGATAA to CGGATCC (i.e. mutant GATA) did
not compete with the binding (Fig. 2, lane 4). This
modification is identical to that used above in the site-directed
mutagenesis analysis in which cadmium-inducible reporter gene
expression did not occur. An oligonucleotide, in which a sequence
adjacent to the GATA1.1 element was modified, did compete with complex
formation (Fig. 2, lane 5). In addition, protein-DNA
complexes were not produced when nematode extract proteins were
incubated with a 32P-labeled oligonucleotide that contained
the mutant GATA1.1 sequence (Fig. 2, lanes 7-9).
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Table II
Oligonucleotides used to characterize C. elegans metallothionein GATA
elements
Mutagenic oligonucleotides used as primers in preparing site-direction
mutations of GATA sequences in mtl-1 and mtl-2
promoter fragments are shown.
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Fig. 2.
Electrophoretic mobility shift analysis of
the GATA1.1 sequence and C. elegans extract
proteins. A 32P-labeled, double-stranded
oligonucleotide probe (38 bp, Tables II and III), which contained an
unmodified (WT Probe, lanes 1-5) or mutated
(Mutant Probe, lanes 6-9) GATA1.1 sequence, was
incubated with 10 µg of C. elegans extract protein. Each
probe was incubated in either the absence (None, lanes
2 and 7) or the presence of a 50-fold molar excess of
unlabeled competitor. Wild type oligonucleotide (WT, lane 3), an oligonucleotide in which the GATA sequence was
changed from CTGATAA to CGGATCC (mut-G; lanes 4 and 8), or an oligonucleotide containing a mutation adjacent
to the GATA sequence (mut-M; lanes 5 and
9) were used. A single, sequence-specific protein-DNA
complex (I) is evident. NE, no extract
(lanes 1 and 6).
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EMSA were also used to examine protein interactions with the GATA2.1
element from mtl-2. A DNA-protein complex was formed between
C. elegans extract proteins and a labeled oligonucleotide that included the GATA2.1 sequence (Fig.
3, lane 2). The formation of
this complex was successfully competed by the addition of excess of
unlabeled oligonucleotide (Fig. 3, lanes 3-5). Addition of up to a 100-fold molar excess of mutant GATA2.1 oligonucleotide (Tables
II and III) did not affect complex formation (Fig. 3, lanes 6-8). Furthermore, a GATA-specific protein-DNA complex was not generated when C. elegans proteins were combined with a
labeled oligonucleotide containing the mutant GATA2.1 sequence (Fig. 3, lanes 10-13). Similar results were obtained when
oligonucleotides that contained the GATA 2.4 sequences were used (data
not shown).
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Fig. 3.
Electrophoretic mobility shift analysis of
the GATA2.1 element and C. elegans extract
proteins. A 32P-labeled, double-stranded wild type
(WT Probe, lanes 1-8) or mutated (Mutant
Probe, lanes 9-13) GATA2.4 oligonucleotide (Tables II
and III) was incubated with 10 µg of C. elegans extract
protein. Unlabeled WT (lanes 3-5) or
Mutant (lanes 6-8, and 10-13)
competitor oligonucleotides were added in 0-100-fold molar excess. A
single sequence specific band (I) is evident only in the
samples containing labeled wild type probe (lanes 2, 3 and
6-8). NE, no extract (lanes 1 and
9).
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These results indicate that C. elegans proteins specifically
bind to mtl-1 and mtl-2 GATA elements. However,
no differences in mobility shift patterns were observed in extracts
prepared from cadmium-treated versus non-treated C. elegans (data not shown).
Binding of ELT-2 to mtl-1 and mtl-2 GATA Elements--
The ability
of the C. elegans GATA-binding transcription factor ELT-2 to
form complexes with GATA1.1, GATA2.1, or GATA2.4 elements was
investigated. Oligonucleotide probes that included these GATA sequences
were incubated with in vitro transcribed/translated ELT-2
protein. ELT-2-DNA complexes were formed in reactions that contained
any of the three oligonucleotide probes (Fig.
4). Formation of these complexes was
successfully competed by the addition of identical, unlabeled
oligonucleotides (Fig. 4, lanes 4-6). In contrast, the
addition of up to 100-fold molar excesses of unlabeled mutant
oligonucleotides, in which GATA sequences were changed from TGATAA to
GGATCC, had no effect on complex formation (Fig. 4, lanes
8-10). These results demonstrate that ELT-2 binds in a
sequence-specific manner to the mtl-1 and mtl-2
GATA elements that were shown essential for transcription.
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Fig. 4.
Binding of in vitro synthesized ELT-2 to mtl-1 and mtl-2 GATA elements. 32P-Labeled, double-stranded,
25-bp oligonucleotide probes (Tables II and III) that included the GATA
1.1 sequence (upper panel), GATA2.1 sequence (middle
panel), or GATA2.4 sequence (lower panel) were
incubated with in vitro transcribed/translated ELT-2
protein. A molar excess of unlabeled, double-stranded wild type or
mutant competitor oligonucleotides were included in the reactions at
the levels indicated.
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In Vivo Activation of mtl-2 Transcription by ELT-2--
Several
strains of C. elegans containing double transgenes were
prepared to define further the interaction between the regulation of
C. elegans MT transcription and GATA factors in
vivo. When C. elegans strain JF5(hsp-16/elt-2,
mtl-2/lacZ) was heat-shocked, -galactosidase expression was
detected in hypodermal, muscle, nerve, and pharyngeal cells of L3-L4
larva and adult nematodes (Fig. 5,
A and B). The mtl-2 gene is not
normally transcribed in these cell types (22). This indicates that
ELT-2 alone is sufficient to induce mtl-2 transcription.
Interestingly, reporter gene expression was not observed in the
intestinal cells of L3-L4 larvae and adult JF5(hsp-16/elt-2,
mtl-2/lacZ) nematodes. Heat-shock induced reporter gene
transcription in the intestinal cells of JF5(hsp-16/elt-2,
mtl-2/lacZ) embryos (2-fold or later) or L1-L2 larva (Fig. 5,
C and D). In contrast, heat-shock did not induce mtl-2 transcription in embryos or non-intestinal cells of
nematodes that did not ectopically express ELT-2 (i.e.
JF4(mtl-2/lacZ)). Furthermore, the frequency and level of
reporter gene expression in the intestinal cells of heat-shocked
JF4(mtl-2/lacZ) C. elegans was 10-fold lower
compared with JF5(hsp-16/elt-2, mtl-2/lacZ). Exposure of the
JF4(mtl-2/lacZ) or JF5(hsp-16/elt-2, mtl-2/lacZ) strains to cadmium induced reporter gene transcription exclusively in
the intestinal cells of post-embryonic nematodes (Fig.
6, A and B).
Activation of mtl-2 transcription did not occur in either strain in the absence of metal or heat-shock (Fig. 6, C
and D).
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Fig. 5.
Transcription of mtl-2 in
C. elegans ectopically expressing ELT-2. C. elegans strain JF5(hsp-16/elt-2, mtl-2/lacZ) was
heat-shocked at 34 °C and then stained for -galactosidase
activity (see "Experimental Procedures"). A shows adult
hermaphrodite nematodes with stained body wall and pharyngeal muscle
and hypodermal cell nuclei. Arrowheads indicate the location
of stained hypodermal and body wall muscle nuclei. B is an
enlargement of a stained adult pharynx. Black arrowheads
indicate the location of stained hypodermal nuclei. White
arrowheads identify stained pharyngeal muscle cell nuclei.
C and D, reveal mtl-2 promoter
activity in the intestinal cells of a late embryo and L1 larva,
respectively. Arrowheads in these two panels
indicate intestinal cell nuclei that are expressing the reporter
transgene. ph signifies the location of the C. elegans pharynx.
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Fig. 6.
Cell-specific expression of the
mtl-2/lacZ transgene. The patterns of
-galactosidase expression in mixed populations of C. elegans strains JF4(mtl-2/lacZ) (A and
C) and JF5(hsp-16/elt-2, mtl-2/lacZ)
(B and D). Nematodes were exposed to 100 µM cadmium for 24 h (A and B)
or non-induced (C and D) prior to staining.
A and B show reporter gene activity exclusively
in intestinal cells of adults and larvae. Arrowheads in
A and B identify some of the intestinal cell
nuclei that are expressing the reporter transgene. ph
signifies the location of the C. elegans pharynx.
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Heat-shock treatment of C. elegans strain
JF6(hsp-16/elt-1 and mtl-2/lacZ), which
ectopically expresses ELT-1, predominantly activated mtl-2
transcription in intestinal cells. The level of intestinal cell
expression was comparable to that of heat-shocked JF4(mtl-2/lacZ) nematodes. Ectopic expression of ELT-1 did
not activate mtl-2 transcription in embryonic intestinal
cells. Reporter gene expression in non-intestinal cells
(i.e. body wall and pharynx muscle and hypodermal cells) was
observed in 1-2% of the -galactosidase-expressing JF6(hsp-16/elt-1, mtl-2/lacZ) C. elegans.
However, the levels and frequency of expression were considerably lower
than those observed in the JF5(hsp-16/elt-2, mtl-2/lacZ)
strain and are not considered significant. These results suggest that
ELT-1 does not contribute to the regulation of C. elegans MT transcription.
An additional strain of C. elegans,
JF7(mtl-2/lacZ,
elt-2/+), was created through a genetic cross
of JF4(mtl-2/lacZ) with the homozygous null
mutant elt-2(ca15) (45). C. elegans
with elt-2/+ genotype was identified by
polymerase chain reaction of genomic DNA isolated from individual
nematodes using primers that flank elt-2 (45). Single
JF7(mtl-2/lacZ,
elt-2/+) nematodes were placed on
NGM agar plates, which contained 100 µM
CdCl2, and allowed to grow for ~3 days. The progeny were
then stained for -galactosidase activity. Of the 357 nematodes
examined, 165 (46%) expressed the reporter gene in the intestinal
cells. These C. elegans were either
elt-2/+ or elt-2+/+, as
determined by the proper development of the intestinal lumen (45) (Fig.
7B). Fifty-seven nematodes
were elt-2/, as determined by the
improperly formed intestine. The majority of these nematodes did not
express -galactosidase (Fig. 7A). Five of the
elt-2/ C. elegans showed reporter gene
expression in one or two intestinal cells. However, the level of
-galactosidase expression was substantially lower than that observed
in elt-2/+ or elt-2+/+
C. elegans and is not significant. When the
JF4(mtl-2/lacZ) strain was grown on
cadmium-containing NGM plates, only ~55% of the progeny expressed
the transgene in the intestinal cells.
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Fig. 7.
Transcription of mtl-2 in
elt-2(ca15) null mutant C. elegans. The progeny from a single
JF7(mtl-2/lacZ+/+,
elt-2/+) nematode were exposed to 100 µM cadmium and then stained for -galactosidase
activity. Nuclear staining of the intestinal cells is not evident in an
elt-2/ nematode (A).
In contrast, in an elt-2± C. elegans
(B) intestinal cells actively transcribe mtl-2.
Both nematodes are L1 larva. Arrowheads in A
identify the intestinal cell nuclei that are expressing the reporter
transgene.
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The ability of ectopically expressed ELT-2 to activate mtl-2
transcription (a) and the lack of mtl-2
transcription in elt-2/ C. elegans (b) indicate that this GATA factor regulates
C. elegans MT transcription in vivo.
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DISCUSSION |
The ability of transition metals, as well as other physiologic
stressors, to induce MT expression has been established for decades
(1). Two models have been proposed that describe how metals induce MT
transcription (25, 26). However, metal exposure alone is not sufficient
to activate expression. For example, cadmium accumulates in the
prostate of metal-exposed rats, but MT expression is not induced in
this tissue (66). Thus, cell-specific processes are also involved in
regulating MT gene expression.
A comparison among the nucleotide sequences of mtl-1 and
mtl-2, and other C. elegans genes whose
expression is limited to intestinal cells, reveals the presence of
multiple potential binding sites for GATA family transcription factors
(Table I). Because GATA elements are involved in the regulation of
cell-specific gene expression in a variety of organisms, they may also
participate in the intestine-specific expression of mtl-1
and mtl-2. In vivo deletion and site-directed mutagenesis
analyses confirm that one of these elements in mtl-1 and two
in mtl-2 are required for cadmium-inducible MT transcription
(Table IV and Fig. 1).
Six families of vertebrate GATA-binding transcription factors, GATA-1
through GATA-6, have been identified. Each has distinct but overlapping
tissue-specific patterns of expression. Members of the vertebrate
GATA-4/5/6 family are expressed in intestinal cells. The factors may be
involved in gastrointestinal development (a) and may
regulate intestinal cell-specific gene expression (67-74)
(b). The DNA binding domain of ELT-2 is most closely related to the vertebrate GATA-5 factor (50).
Four GATA factors have been isolated from C. elegans: ELT-1,
ELT-2, ELT-3, and END-1. The expression of ELT-1 and ELT-3 is limited
to embryonic epidermal and hypodermal cells, respectively (51, 52). The
GATA factor END-1 is involved in the development of the nematode
intestine. C. elegans intestinal cells arise from a single
progenitor cell, E cell, which is formed at the 8-cell stage of
embryogenesis (75). END-1 is only detected between the 1E and 4E cell
stages of development and functions during early intestine formation
(49). The pattern of end-1 expression does not overlap those
of C. elegans MT genes. Thus, ELT-1, ELT-3, and END-1 are
not likely to be regulators of C. elegans MT transcription.
In contrast, Elt-2 protein and mRNA are first detected at the
2E-cell stage and are present in all of the intestinal cells. They are
continuously expressed throughout development and in the adult nematode
(50). The post-embryonic pattern of elt-2 expression is
identical to those of mtl-1 and mtl-2
(i.e. exclusively in intestinal cells), which suggests that
it may be a regulator of MT gene expression.
Electrophoretic mobility shift assays, using C. elegans
protein extracts, confirms that nematode protein(s) bind to the
mtl-1 and mtl-2 GATA elements, which were shown
by in vivo mutagenesis analysis to be required for MT
transcription (Figs. 2 and 3). In vitro expressed ELT-2
forms a sequence-specific complex with these GATA elements (Fig. 4).
These results confirm that ELT-2 binds to the C. elegans MT
GATA elements. However, they do not conclusively demonstrate that ELT-2
regulates MT transcription.
Ectopic expression of ELT-2 activates mtl-2 transcription in
the absence of metal exposure (Fig. 5, A-D).
This result indicates that ELT-2 regulates C. elegans MT
transcription. Similar results were obtained in studies of the gut
esterase gene, ges-1. Embryonic expression of GES-1 is
limited to the E cell lineage (42, 46). When ELT-2 is ectopically
expressed, however, GES-1 is observed in most of the cells in the
embryo (45).
Heat-shock of JF5(hsp-16/elt-2, mtl-2/lacZ) nematodes
activates mtl-2 transcription in intestinal cells of embryos
at later stages of development (Fig. 5C) (a);
intestinal cells of L1-L2 larvae (Fig. 5D) (b);
and hypodermal, pharynx, muscle, and nerve cells of L3-L4 larvae and
adults (Fig. 5, A and B) (c). The
pattern, level, and frequency of mtl-2 transcription in
nematodes that express ELT-2 in all cell types are significantly
different than those observed in cadmium-treated animals. In addition,
heat-shock of the JF4(mtl-2/lacZ) strain does not induce
significant levels of mtl-2 transcription. These results
indicate that ELT-2 alone is sufficient to activate MT transcription,
i.e. elevated concentrations of metal are not necessary to
induce transcription.
Some characteristics of GATA elements and ELT-2 resemble those of MREs
and MTF-1. For example, deletion or modification of specific
GATA-sequences in mtl-1 or mtl-2 prevents
metal-inducible transcription in vivo (Fig. 1). Likewise,
deletion or modification of specific MREs will prevent metal-inducible
transcription of mammalian MTs (76). However, data indicate that GATA
elements and ELT-2 do not control metal responsiveness. GATA elements
and ELT-2 are responsible for determining the intestinal cell
specificity of MT transcription. GATA elements are found in the
upstream regulatory regions of C. elegans intestinal
cell-specific genes including vitellogenins, gut esterase,
P-glycoprotein, and cysteine protease (41, 44, 46, 77). Metal-inducible
transcription of these genes has not been reported, suggesting that
GATA sequences do not function as C. elegans MREs. In
addition, cadmium exposure does not induce the transcription of a GATA
element containing intestinal cell-specific aspartic
protease.2 ELT-2 contains a
single zinc finger domain; however, mouse and human MTF-1 contain
multiple zinc fingers (25, 50). A characteristic of MTF-1, and other
mammalian metal-responsive transcription factors, is the ability of low
concentrations of EDTA to inhibit MTF-1-DNA complex formation (25,
78-80). The binding of ELT-2 to GATA elements is significantly less
sensitive to EDTA than MTF-1 binding to MREs.3
The transcription of C. elegans MT genes is limited to
intestinal cells as a consequence of the presence of GATA elements in
the promoters (a) and ELT-2 being expressed exclusively in intestinal cells (b). Thus, intestinal cell-specific
expression is controlled simply by limiting the expression of an
essential transcription factor to this specific cell lineage. ELT-2 can activate MT transcription in the absence of metal exposure (Fig. 5),
and it is constitutively expressed in intestinal cells in embryonic and
post-embryonic stage of development (45, 50). However, mtl-1
and mtl-2 are not usually transcribed unless the nematodes
are exposed to metal. This indicates that additional regulatory
processes contribute to the control of metal-inducible MT gene
expression. Since ELT-2 is not the "metal sensor," additional factors must act to regulate the metal inducibility. These factors may
directly interact with ELT-2. Vertebrate GATA-factors interact with a
variety of proteins, including AP-1, SP-1, and YY-1 (81-87). This
interaction can result in either the stimulation or repression of
transcription. Alternatively, UREs that bind metal-responsive transcription factors independently of GATA factors may be present in
the promoters of the C. elegans MT genes.
ELT-2 expression in embryos failed to induce mtl-2
transcription in intestinal cells before the 2-fold stage of
development. Previous studies have shown that heat-shocked
JM57(hsp-16/elt-2) express ELT-2 protein in most of the
cells in early embryos. Ectopically expressed ELT-2 induces the
expression of other intestinal cell-specific genes in non-intestinal
cell lineages (45). Thus, although ELT-2 alone appears to be sufficient
to active mtl-2 transcription, additional processes must
participate in determining the correct developmental pattern of
expression. Developmentally modulated MT expression has been described
in a variety of other species. Transcription of the Drosophila
Mtn gene is not detected until the beginning of germ band
retraction in endodermal gut primordia (88). In mice, MT-IV is not
detected prior to day 7 postpartum (17). Furthermore, MT is not
detected in human fetal brain in less than the 35-week-old fetus (89).
The mechanisms responsible for developmental regulation of MT
transcription have not been elucidated.
Several observations are consistent with a model for the regulation of
metal-inducible, intestinal cell-specific C. elegans MT gene
expression that incorporates a metal-sensitive repressor protein.
First, intestinal cell expression of mtl-2 in heat-shocked L3-L4 and adult JF5(hsp-16/elt-2, mtl-2/lacZ) nematodes is
infrequent and weak compared with other cell types and the levels
observed in cadmium-treated nematodes. Second, although ELT-2 alone
activates MT transcription independent of cell type or metal exposure,
and it is constitutively expressed in the intestinal cells, the MT genes are only transcribed following metal exposure. In the model, a
repressor protein inhibits the ability of ELT-2 to constitutively activate MT transcription. Since cadmium treatment did not affect the
binding of ELT-2 to the GATA elements, the metalloregulatory protein
would not inhibit ELT-2 binding, rather it prevents ELT-2-mediated transcriptional activation of the C. elegans MT genes. In
the presence of metal, repression is released and then ELT-2 can
activate transcription in the intestine.
Repressor-mediated regulation of stress-inducible gene transcription
has been described for several genes. Palmiter (26) has hypothesized
that a zinc-sensitive inhibitor blocks MTF-1 binding to MREs to prevent
MT transcription. It has been reported that overexpression of the p80
subunit of the protein Ku suppresses metal-inducible MT-I gene
transcription in Rat 1 fibroblast cells. The p80 subunit does not
directly inhibit MT-I transcription, but its overexpression may
activate other factors that inhibit transcription (90). The
transcription factor NF
B participates in transcriptional control of
genes responsive to oxidative stress (91). In the absence of stress,
NFB DNA binding is repressed by the inhibitory factor IB, which
forms a complex with NFB. Treatment with pro-oxidants results in the
dissociation of the IB from NFB and the subsequent activation of
target genes. A mammalian heat-shock factor-1 (HSF1)-binding protein
has been identified, HSBP1, that directly interacts with the HSF1.
HSBP1 inhibits the binding of HSF1 to heat-shock promoter elements to repress heat-shock protein transcription (92). The overexpression of
the C. elegans HSBP1 homologue, HSB-1, both inhibits
hsp-16 transcription and reduces the survival of
heat-stressed nematodes (92).
The ectopic expression of ELT-2 in late embryos and L1-L2 larvae
exclusively activates mtl-2 transcription in intestinal
cells. The lack of expression in non-intestinal cells may be due to the presence of developmental stage-specific regulators, which are not
present in older larvae and adult C. elegans. The intestinal expression may be the result of relatively high levels of ELT-2 produced in the heat-shocked JF5(hsp-16/elt-2, mtl-2/lacZ)
nematodes that may overcome or "titrate out" a repressor protein.
Further investigations will be necessary to confirm the repressor model and resolve any inconsistencies.
Regulation of gene expression by GATA elements and transcription
factors is an evolutionarily conserved process. Since ELT-2, a
homologue of vertebrate GATA (50), can bind to mtl-1 and
mtl-2 UREs and control MT transcription, a similar process
may function in higher eukaryotes. Sequence analysis of the upstream
regulatory regions in MT genes, from a variety of species, identified
multiple copies of the GATA consensus sequence. The GATA sequences are interspersed among consensus MRE sequences in invertebrates (sea urchin
and fly), amphibians (frog), fishes (stone loach, rainbow trout,
northern pike, and carp), and mammals (rat, mouse, Chinese hamster,
sheep, and human). The functionality of GATA elements and transcription
factors in regulating the expression of these MT genes has not been
examined. GATA elements are also present in several invertebrate MT
genes that show highly restricted patterns of expression, including the
Drosophila Mto gene (4, 20) and sea urchin spMTA
and spMTB genes (18, 19, 93). In addition, the expression
patterns of the Drosophila GATA factors "serpent" and
dGATA-c overlap those for Mtn and Mto (71, 94,
95). Thus, GATA elements and factors may be components of a
evolutionarily conserved mechanism that controls cell-specific
transcription of MT genes.
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ACKNOWLEDGEMENTS |
We thank Dr. Jim McGhee, University of
Calgary, Alberta, Canada, for the ELT-2 cDNA clone and helpful
discussions. Several nematode strains were obtained from the
Caenorhabditis Genetic Center, funded by the National
Institutes of Health.
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FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants CA 61337 (to J. H. F.) and by the Alberta Heritage Foundation for Medical Research (to T. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: ISSI Consulting Group, Inc., 999 18th St., Suite
1450, Denver, CO 80202.
**
To whom correspondence should be addressed: Box 90328, Duke
University, Durham, NC 27708-0328. Tel.: 919-613-8037; Fax:
919-684-8741; E-mail: jonf@duke.edu.
2
J. H. Freedman, unpublished observations.
3
J. H. Freedman and L. H. Moilanen,
unpublished observations.
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ABBREVIATIONS |
The abbreviations used are:
MT, metallothionein;
bp, base pair(s);
EMSA, electrophoretic mobility shift assay;
MRE, metal-responsive element;
URE, upstream regulatory element;
kbp, kilobase pair.
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REFERENCES |
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ABSTRACT
INTRODUCTION
