 |
INTRODUCTION |
Regulation of the expression of many genes is mediated by the
binding of transcription factors to cis-elements in their
promoter regions. The promoter regions of many eukaryotic genes contain GC-rich sequences (1) and some of the most widely distributed promoter
elements are GC boxes and related motifs. The zinc finger proteins Sp1
and MAZ1
(Myc-associated zinc finger
protein) are transcription factors that bind to GC-rich
sequences, namely GGGCGG and GGGAGGG, respectively, to activate the
expression of various target genes.
Sp1 was originally characterized as a ubiquitous transcription factor,
consisting of 778 amino acids, that recognized GC-rich sequences in the
early promoter of simian virus 40 (2, 3). The DNA-binding domain of Sp1
consists of three contiguous C2H2-type zinc fingers (4). The
amino-terminal region contains two serine- and threonine-rich domains
and two glutamine-rich domains, which are essential for transcriptional
activity (5). The carboxyl-terminal domain of Sp1 is involved in
synergistic activation and interactions with other transcription
factors. Sp1 is considered to be a constitutively expressed
transcription factor and has been implicated in the regulation of a
wide variety of housekeeping genes, tissue-specific genes, and genes
involved in the regulation of growth (6). Sp1 is a phosphorylated (7)
and highly glycosylated protein (8). It interacts with many factors,
such as the TATA box-binding protein, which is a major component
of the general transcription machinery, and the TATA box-binding
protein-associated factors dTAFII110 (9), hTAFII130 (10), and hTAFII55
(11). Other proteins, such as transcription factor YY1 (12, 13), E2F
(14, 15), and p300 (16, 17), have also been reported to associate with
Sp1. Sp1-null mice embryos exhibited severely retarded growth and died
within 10 days (18), after displaying a wide range of abnormalities.
Some of the embryos appeared as an undifferentiated mass of cells,
whereas others had all the typical hallmarks of early embryogenesis,
such as a developing heart, eyes, optic vesicles, somites, erythroid
cells, and extra-embryonic tissues (18). Thus, it is likely that Sp1 is
essential for the differentiation of embryonal stem cells after day 10 of development.
MAZ was first identified as a transcription factor that bound to a GA
box (GGGAGGG) at the ME1a1 site of the c-myc promoter and to
the CT element of the c-myc gene (19-21). It is a zinc
finger protein with six C2H2-type zinc fingers at the carboxyl
terminus, a proline-rich region, and three alanine repeats. It is
expressed ubiquitously, albeit at different levels in different human
tissues (22). It can regulate the expression of numerous genes, such as
c-myc (19, 20, 23, 24), genes for insulin I and II (25), the
gene for CD4 (26), the gene for the serotonin receptor (27), and the
gene for nitric-oxide synthase (28). MAZ might be involved in the
termination of transcription by interrupting elongation by RNA
polymerase II (29).
The promoter region of the gene for MAZ has features
typical of the promoter of a housekeeping gene, namely a high G+C
content, a high frequency of CpG (where p stands for "phosphoric
residue") dinucleotides, the absence of canonical TATA boxes,
and multiple sites for initiation of transcription (30, 31). Moreover, the gene is ubiquitously expressed in human tissues (22).
We have attempted to clarify some aspects of the relationship between
the factors that bind to GC-rich cis-elements and the promoters of housekeeping genes with a high G+C content. A previous study showed that Sp1 binds to GC-rich DNA sequences in nucleosomes (32). Moreover, the large coactivator complex known as CRSP (cofactor
required for activation of Sp1) stimulates Sp1-mediated transcription
(33). Both Sp1 and MAZ can exert positive and negative control over the
expression of target genes. Thus, regulation by individual DNA-binding
factors seems to be coordinated via recruitment of other factors that
participate in the regulated expression of target genes and via
recognition of the modification of nucleotide sequences, for example,
by methylation or demethylation and acetylation or deacetylation
(34-37). The binding affinities of transcription factors for
individual target sequences are likely to be essential parameters in
the regulation of gene expression, together with the recruitment of
related factors.
We demonstrate here a possible mechanism for regulation of the
expression of the human gene for MAZ. The mechanism involves the
recruitment of different repressors by two different DNA-binding factors, Sp1 and MAZ, that interact with the same
cis-elements. Our results indicate that deacetylation and
methylation might be involved in the regulation of a single gene via
the binding of different zinc finger proteins.
 |
MATERIALS AND METHODS |
Plasmids--
A series of DNA fragments from the MAZ promoter
was excised with appropriate restriction enzymes. Each fragment was
filled in and inserted into the HindIII site of pSV00CAT
(38), via a HindIII linker, to generate pMAZCAT1, pMAZCAT2,
pMAZCAT3, pMAZCAT4, and pMAZCAT5, respectively. Internal deletion
mutants of the MAZ promoter were created by amplification by the
polymerase chain reaction, ligation of the appropriate DNA fragments,
and insertion into the HindIII site of pSV00CAT to generate
pMAZCAT2-d, pMAZCAT3-wt, pMAZCAT3-
I, pMAZCAT3-
II, and
pMAZCAT3-
III, respectively. Mutant forms of pMAZCAT3 were further
generated by mutation of dinucleotides (AA to GG; TT to GG (see Fig.
2)) to generate a series of mutants, pMAZCAT3-f1-pMAZCAT3-f11.
Mutations in the putative Sp1-binding sites and putative MAZ-binding
sites in pMAZCAT3-wt were generated by converting the GC-rich motif
GGGCGG to GGTTGG and the GC-rich motif GGGAGGG to GGTATGG (39, 40).
Amplification by polymerase chain reaction and ligation into the
HindIII site of pSV00CAT generated pMAZCAT3-m1-pMAZCAT3-m8.
pCMV-MAZ was constructed as described previously (22). pCMV-Sp1 and
pCMV-DNMT1 were provided by R. Chiu and R. Raenish, respectively.
Cell Culture, Transfection, and Assay of Chroramphenicol
Acetyltransferase (CAT) Activity--
HeLa cells, 293 cells, and
NIH3T3 cells were grown in Dulbecco's modified Eagle's medium that
had been supplemented with 10% fetal bovine serum (Life Technologies,
Inc.). NCI-H460 cells were grown in RPMI 1640 medium that had
been supplemented with 10% fetal bovine serum. Cells were treated with
tricostatin A (TSA) at a final concentration of 100 ng/ml and with
5-azacytidine at a final concentration of 1 mM. Cells were
transfected with plasmid DNA using the FuGENETM 6 transfection reagent (Roche Molecular Biochemicals) according to the
protocol provided by the manufacturer. All plasmids were purified by
ultracentrifugation before transfection, as described previously (41).
Assays of CAT activity were performed as described elsewhere (21).
Gel Shift Assay--
DNA probes were radiolabeled at their
5'-ends with polynucleotide kinase (New England BioLabs, Inc., Beverly,
MA) and [
-32P]ATP. The DNA probes designated M, S, and
MS corresponded to DNA fragments between nt
313 and
284, nt
232
and
216, and nt
151 and
137. The binding reaction was performed
in 30 µl of a buffer that contained 20 mM Tris-HCl (pH
7.5), 2 mM MgCl, 0.5 mM EDTA, 10% glycerol,
0.5 mM dithiothreitol, 25 mM NaCl, 1 µg of
poly(dI-dC), and an extract of HeLa cells or purified glutathione
S-transferase (GST) fusion proteins. Reactions were incubated at 4 °C for 40 min after addition of the labeled DNA probe. The incubation was continued for 30 min at room temperature after the addition of appropriate antibodies. Products of reactions were loaded onto a 5% non-denaturing polyacrylamide gel in 0.5× TBE buffer (1× TBE: 45 mM Tris-borate, 1 mM EDTA). Electrophoresis was performed at 100 V for 4-6 h
at 4 °C.
Immunoprecipitation-Western Blotting
Analysis--
Immunoprecipitation- Western blotting analysis was
performed as described previously (22).
Immunoprecipitation and Assay of Histone Deacetylase (HDAC)
Activity--
HeLa cells were cultured with or without TSA (100 ng/ml)
for 48 h, and then proteins in cell extracts were
immunoprecipitated with antibodies specific for HDACs (Santa Cruz
Biotechnology, Santa Cruz, CA) or DNA methyltransferase 1 (DNMT1) (New
England BioLabs, Inc.). Cell extracts were subjected to assays
of HDAC activity using a histone deacetylase assay kit (Upstate
Biotechnology, Lake Placid, NY) in accordance with the instructions
from the manufacturer.
 |
RESULTS |
Unique Symmetric Elements in the Minimal MAZ Promoter Are Essential
for Transcriptional Activity--
The promoter region of the human
gene for MAZ has an extremely high G+C content, namely 88.4%. Various
GC-rich elements are present in the promoter region, including
consensus Sp1-binding sites (GGGCGG) and consensus MAZ-binding sites
(GGGAGGG) (Fig. 1). Some of these sites
overlap one another. Assays of CAT activity using constructs with
various deletions in the MAZ promoter demonstrated that the minimal
promoter activity was localized between nt
383 and +259 (Fig.
2A). Internal deletion of the
region between nt
383 and
248 (pMAZCAT2-d) resulted in a decrease
in promoter activity. This result suggested that the region from nt
383 to
248 might be critical for minimal promoter activity. We next attempted to identify the elements that were essential for minimal promoter activity. Promoter activity was reduced with the construct that lacked the region between nt
383 and
334, whereas constructs with internal deletion of the region between nt
334 and
279 or
between nt
279 and
248 did not have reduced promoter activity (Fig.
2B). Thus, the region from nt
383 to
334 was essential for the promoter activity. Four symmetric elements, namely two CAAC
and two CTTC elements, were present in this region (Fig. 2C). CAAC and CTTC elements have also been found in other
promoters, such as the promoter of the gene for the
-myosin heavy
chain (42), the gene for hydroxymethylbilane synthase (43), the gene
for myosin light chain 2 (44), and the gene for lactoferrin (45). Some
of these sites have been shown to contribute to the activation of
promoter activity. We used a series of CAT constructs with mutations in
these elements to investigate whether these putative elements might
activate the promoter of the gene for MAZ (Fig. 2D). The
results of CAT assays demonstrated that promoter activity was reduced
when each of the four elements was mutated (pMAZCAT3-f1,
pMAZCAT3-f2, pMAZCAT3-f3, and pMAZCAT3-f4). The promoter
activity was reduced still further when two of the four elements were
mutated simultaneously (pMAZCAT3-f5, pMAZCAT3-f6, pMAZCAT3-f7,
pMAZCAT3-f8, pMAZCAT3-f9, and pMAZCAT3-f10). The CAT activity
fell to about 20% of that of the wild type when all of the four
elements were mutated (pMAZCAT3-f11). These results indicated that the
four symmetric elements were essential for the activity of the MAZ
promoter.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 1.
The GC-rich promoter of the gene for MAZ
contains multiple putative DNA-binding sites for both Sp1 and MAZ.
The putative Sp1-binding sites and MAZ-binding sites are indicated by
wavy underlining and boxes, respectively. The
sequence is numbered with the major site of initiation of transcription
taken as +1.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
A symmetric element in the minimal MAZ
promoter is essential for the promoter activity of the gene for
MAZ. A, the minimal basal promoter of the gene for MAZ
was located in the region between nt 383 and +259. A summary is shown
of the human MAZ-CAT deletion constructs and corresponding CAT
activities. Numbering is relative to the major site of initiation of
transcription (+1). CAT, gene for CAT. CAT fusion plasmids
were used to transfect HeLa cells, and CAT activity was measured as
described under "Materials and Methods." Promoter activities of
MAZ-CAT fusion genes are expressed relative to the activity of
pMAZCAT1, which was taken arbitrarily as 1.0. All values in this and
other figures are the averages of results from at least three
experiments, and the standard deviation for each value is indicated.
B, the region between nt 383 and 334 is critical for the
promoter activity of the MAZ gene. Promoter activities of MAZ-CAT fusion genes are expressed relative to the activity of pMAZCAT3,
which was taken arbitrarily as 1.0. C, unique symmetric
elements were present in the region between nt 383 and 334.
Boxes indicate four dinucleotide repeats. D, the
symmetric elements are essential for the promoter activity of the gene
for MAZ. The wild type CAAC and CTTC elements and the mutated element
CGGC were examined for their effects on the promoter activity of the
gene for MAZ. Promoter activities of MAZ-CAT fusion genes are expressed
relative to the activity of pMAZCAT3, which was taken arbitrarily as
1.0.
|
|
Sp1 and MAZ Recognize the Same cis-Elements in the MAZ
Promoter--
To determine whether Sp1 and/or MAZ could bind to the
various putative binding sites for both Sp1 and MAZ, we performed gel shift assays using extracts of HeLa cells and DNA probes derived from
the MAZ promoter. The SM probe, nt
313 to
284, contained one
putative Sp1-binding site and one putative MAZ-binding site, and these
two sites partially overlapped. The M probe, nt
232 to
216,
contained one putative MAZ-binding site; and the S probe, nt
153 to
137, contained one putative Sp1-binding site (Fig. 3A). We detected two prominent
DNA-protein complexes with the SM probe that contained the overlapping
binding sites for Sp1 and MAZ. The rapidly migrating band was more
intense than the slowly migrating band (Fig. 3B, left
panel). The retarded bands corresponding to B1 and B2 were shifted
even further upon addition of antibodies against Sp1 and MAZ (Fig.
3B, lanes 2 and 4). Control antibodies did not affect the mobility of the DNA-protein complexes (Fig. 3B, lane 6). These results indicated that
both Sp1 and MAZ specifically recognized the overlapping sites in the
same cis-element. To examine the DNA binding specificity of
Sp1 and MAZ, we used the purified GST-Sp1 and GST-MAZ in the assays. A
DNA-protein complex was detected using GST-Sp1, and a supershifted band
was detected in the presence of antibodies specific for Sp1 but not in
the presence of antibodies specific for MAZ. Similarly, a DNA-protein complex was detected using GST-MAZ, and a supershifted band was detected in the presence of antibodies to MAZ but not in the presence of antibodies to Sp1 (Fig. 3B, lanes 7-12).
Supershifted bands were also detected in the presence of antibodies
against MAZ or Sp1 when we used the M probe or the S probe (Fig.
3C, lanes 2, 4, 8, and
10). The results indicated that both Sp1 and MAZ interacted with the putative MAZ-binding sites and the putative Sp1-binding sites
in all three probes that we used. We also examined other GC-rich
cis-elements in the MAZ promoter for recognition by Sp1 and
MAZ. We found that Sp1 and MAZ bound to the same GC-rich
cis-elements in other regions of the MAZ promoter (data not
shown). Thus, it was clear that Sp1 and MAZ bound to the same GC-rich
cis-elements in the MAZ promoter.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 3.
Sp1 and MAZ bind to the same DNA-binding
sites in the promoter of the gene for MAZ. A, the
nucleotide sequences of the DNA probes in the promoter region are
shown, and the binding sites for Sp1 and MAZ are indicated.
B and C, both Sp1 and MAZ bind to the same
consensus sites for Sp1 and MAZ in the MAZ promoter. Shifted
protein-DNA complexes are indicated by closed arrowheads.
Supershifted complexes obtained with antibodies specific for Sp1
( -Sp1) and MAZ ( -MAZ), respectively, are indicated by open
arrowheads.
|
|
Both Sp1 and MAZ Repress the Activity of the MAZ Promoter through
the Various cis-Elements--
We next focused on the effects of Sp1
and MAZ in transactivation of the gene for MAZ. The
reporter constructs were used to transfect HeLa cells in the presence
or absence of an Sp1 or a MAZ expression vector. The promoter activity
was inhibited significantly in the presence of ectopically expressed
Sp1 (Fig. 4A) and MAZ (Fig.
4B), whereas ectopic expression of Sp1 and of MAZ had no effect on the transcription of pRSVCAT (19), the control plasmid. These
results indicated that both Sp1 and MAZ repressed transcription of
the gene for MAZ.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Sp1 and MAZ repress the activity of the
MAZ-CAT reporter gene. A, Sp1 repressed the promoter
activity of the gene for MAZ. Transfections and CAT assays were
performed using HeLa cells in the presence and absence of pCMV-Sp1.
Promoter activities of MAZ-CAT fusion genes are expressed relative to
the activity of pMAZCAT1 in the absence of pCMV-Sp1, which was taken
arbitrarily as 1.0. B, MAZ repressed the promoter activity
of the gene for MAZ. Transfections and CAT assays were performed with
HeLa cells in the presence and absence of pCMV-MAZ. Promoter activities
of MAZ-CAT fusion genes are expressed relative to the activity of
pMAZCAT1 in the absence of pCMV-MAZ, which was taken arbitrarily as
1.0.
|
|
The binding sites for Sp1 and MAZ in the minimal promoter region
(-303 to +3) were mutated in an attempt to identify the
cis-elements that were involved in the negative
regulation (Fig. 5). In the presence
of ectopically expressed Sp1 or MAZ, repression of the MAZ promoter was
detected in the presence of mutations in the region between nt
383
and
248 (pMAZCAT3-m1 and pMAZCAT3-m4). We then mutated other sites
(pMAZCAT3-m2, pMAZCAT3-m3, pMAZCAT3-m5, pMAZCAT3-m6, and pMAZCAT3-m7),
and again we observed reduced transcriptional activity. All the mutated
constructs mentioned above contained wild type binding sites for Sp1
and/or MAZ. Thus, those binding sites for Sp1 and/or MAZ might still
have been active in the negative regulation of the MAZ promoter. Our
results indicated that most, if not all, of the binding sites for Sp1
and MAZ were involved in negative regulation of the expression of the
gene for MAZ. This possibility was confirmed by studies of promoter
activity with pMAZCAT3-m8, in which all the binding sites for both Sp1 and MAZ had been mutated. No repression by Sp1 or by MAZ was observed with pMAZCAT3-m8. These results strongly suggested that repression by
Sp1 and/or MAZ was mediated by the DNA-binding sites for Sp1 and MAZ
and that most or all of these sites were involved in the repressive
activity.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5.
Repression is mediated by binding sites for
Sp1 and MAZ in the MAZ promoter. A summary is shown of the wild
type and mutated binding sites for Sp1 and MAZ in the MAZ promoter,
with corresponding CAT activities. Cotransfections and CAT assays were
performed with HeLa cells in the presence and absence of pCMV-Sp1 and
pCMV-MAZ, as described under "Materials and Methods." Promoter
activities of MAZ-CAT fusion genes are expressed relative to the
activity of pMAZCAT3-wt, which was taken arbitrarily as 1.0.
|
|
Independent Repression by Sp1 and by MAZ Is Mediated by Their
Respective Repression Domains--
Sp1 and MAZ repressed the
expression of the gene for MAZ by binding to the same
cis-elements. Therefore, we next asked whether repression by
Sp1 and by MAZ might be related. The results of a yeast two-hybrid
assay and immunoprecipitation-Western blotting analysis showed that Sp1
did not interact with MAZ (data not shown), indicating that repression
by Sp1 and repression by MAZ were independent.
A series of Sp1 and MAZ expression plasmids was constructed to identify
the domains responsible for repression. These plasmids were used to
cotransfect HeLa cells in combination with the reporter construct
(pCATMAZ1), and then CAT assays were performed (Fig. 6, A and B).
Repression of CAT activity was observed with constructs that did not
encode domains in the amino-terminal region of Sp1 (amino acid
positions 1-503; Fig. 6A). These results suggested that the
amino-terminal region of Sp1 was not involved in repression. The
promoter activity was released from repression when Sp1 without the
carboxyl-terminal region (
622-C) was expressed. Moreover, repression of the promoter activity was diminished when Sp1 was expressed without the zinc finger domain (
531-605) that is
essential for binding to DNA. Taken together, the results indicated
that the carboxyl-terminal region of Sp1 (amino acids 622-778) was responsible for the repression and that the repression was also dependent on the DNA binding ability of Sp1.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
Identification of the repression domain of
Sp1 and of MAZ. A, identification of the repression
domain of Sp1. The structures of the various derivatives of Sp1 are
illustrated schematically. ST, serine- and threonine-rich
domain; Q, proline-rich domain; Fingers, zinc
finger domains. The activities of the MAZ promoter are expressed
relative to the activity of the control vector pcDNA 3, which was
taken arbitrarily as 1.0. B, identification of the
repression domain of MAZ. The structures of the various derivatives of
MAZ are illustrated schematically. P-rich, proline-rich
domain. The activities of the MAZ promoter are expressed relative to
the activity of the control vector pcDNA 3, which was taken
arbitrarily as 1.0.
|
|
The promoter activity was partially repressed when MAZ without amino
acids 54-195 (
54-195) was expressed, and the activity of the
promoter was completely released from repression when MAZ without amino
acids 127-292 (
127-292) was expressed. Thus, it appeared that the
region between amino acids 127 and 292 was responsible for repression
of the expression of the gene for MAZ. Moreover, repression of the
promoter was reduced when a mutant form of MAZ was expressed without
the five zinc fingers in the carboxyl-terminal region (
317-441),
which was essential for DNA binding activity. Taken together, the
results demonstrated that amino acids 127-292 of MAZ were responsible
for autorepression and that autorepression was also dependent on the
DNA binding activity of MAZ. We concluded that independent
repression by Sp1 and by MAZ was mediated by the repression domains of
each protein and that the DNA binding activities of these zinc finger
proteins were also essential for repression.
Recruitment of Histone Deacetylases by MAZ--
HDACs are known to
act as repressors in the regulation of the expression of many genes. We
attempted to determine whether histone deacetylases might be involved
in repression of the gene for MAZ. HeLa cells were transfected with
pCMV-MAZ or pCMV-HDAC1 in the presence and absence of TSA, a specific
inhibitor of histone deacetylases. We then monitored the CAT activity
due to a reporter plasmid, pMAZCAT1, with which the cells had been
cotransfected. Ecpotic expression of HDAC1 repressed the activity of
the MAZ promoter, and such repression was overcome in the presence of TSA (Fig. 7A), indicating that
histone deacetylases might be involved in repression by MAZ. This
possibility was confirmed by measurement of the HDAC activity of
proteins that were recruited by MAZ. The HDAC activity of a
MAZ-specific immunoprecipitate was more than five times higher than
that of the complex that was immunoprecipitated by the control IgG, and
the activity of the former complex was repressed in the presence of TSA
(Fig. 7B). We performed immunoprecipitation and Western
blotting analysis using nuclear extracts from HeLa cells to determine
whether histone deacetylases were included in the complex of proteins
recruited by MAZ. Western blotting analysis indicated the presence of
MAZ in immunoprecipitates of extracts obtained with antibodies specific
for HDAC1, HDAC2, and HDAC3 (Fig. 7C). The proteins in the
same extracts were also immunoprecipitated by antibodies specific for
MAZ, and all three kinds of histone deacetylase were detected (Fig.
7C). These results implied that MAZ recruited proteins that
included HDAC1, HDAC2, and HDAC3.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 7.
Recruitment of HDACs by MAZ.
A, involvement of HDACs in repression by MAZ. Transfections
and CAT assays were performed using HeLa cells in the presence and
absence of pCMV-MAZ and pCMV-HDAC1. Cells were collected 48 h
after treatment with or without TSA. Promoter activities of MAZ-CAT
fusion genes are expressed relative to the activity of pMAZCAT1 in the
absence of pCMV-MAZ, which was taken arbitrarily as 1.0. B,
HDAC assays of the complex of proteins that were recruited by MAZ. HeLa
cells were treated with or without TSA for 48 h. Then proteins in
cell extracts were immunoprecipitated with antibodies against MAZ and
assayed for HDAC activity. Mouse IgG was used as the negative control.
C, immunoprecipitation-Western blotting analysis. Proteins
in extracts of HeLa cells were immunoprecipitated with antibodies
against MAZ, HDAC1, HDAC2, and HDAC3, and then Western blotting
analysis was performed. Mouse IgG was used as the negative
control.
|
|
Association of DNMT1 with Repression by Sp1--
HeLa cells were
transfected with pCMV-Sp1 and pMAZCAT1 in the presence or absence of
pCMV-HDAC1 and TSA, respectively. The results of CAT assays revealed
that repression by Sp1 was insensitive to TSA and that ectopic
expression of HDAC1 had no effect on repression by Sp1 (Fig.
8A), suggesting that
repression by Sp1 might be HDAC-independent. Methylation is known to be
important in the regulation of gene expression. Thus we examined
whether methylation might be involved in repression by Sp1. HeLa cells
were transfected with pCMV-Sp1 (or just with the reporter) in the
presence or absence of 5-azacytidine, a specific inhibitor of
methylation. Repression by Sp1 was released in the presence of
5-azacytidine using the wild type reporter but not the mutant reporter
(pMAZCAT3-m8) and the control reporter, pRSVCAT (Fig. 8B).
Furthermore, the forced expression of DNMT1 enhanced the repression of
transcription by Sp1, whereas treatment with 5-azacytidine reversed the
repression due to Sp1 and DNMT1 (Fig. 8C). We performed
immunoprecipitation and Western blotting analysis using nuclear
extracts from HeLa cells, and the results showed that DNMT1 was
included in the complex of Sp1 and vice versa (Fig. 8D).
Taken together, these results suggest that DNMT1 might be involved in
the repression mediated by Sp1.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Association of DNMT1 with repression by
Sp1. A, repression by Sp1 was independent of HDAC1.
Transfections and CAT assays were performed using HeLa cells in the
presence and absence of pCMV-Sp1 and pCMV-HDAC1. The cells were
harvested after a 48-h treatment with TSA. Promoter activities of
MAZ-CAT fusion genes are expressed relative to the activity of
pMAZCAT3-wt in the absence of pCMV-Sp1, which was taken arbitrarily as
1.0. B, repression by Sp1 was sensitive to 5-azacytidine
(5-aza). HeLa cells were transfected with pMAZCAT3-wt,
pMAZCAT3-m8, and the control, pRSVCAT, and then stable clones were
treated with 5-azacytidine for 72 h before assays of CAT activity.
C, transfected HeLa cells with pMAZCAT3-wt were transfected
with pCMV-Sp1 or pCMV-DNMT1 and incubated with or without 5-azacytidine
for 72 h before assays of CAT activity. D,
immunoprecipitation-Western blotting analysis. Proteins in extracts of
HeLa cells were immunoprecipitated with antibodies against Sp1 or
DNMT1, and then Western blotting analysis was performed. Mouse IgG was
used as the negative control.
|
|
 |
DISCUSSION |
The promoter regions of human housekeeping genes are usually
GC-rich, and, by definition, these genes are expressed ubiquitously, as
is, for example, the human gene for MAZ (22, 30). Many GC-rich
cis-elements can be found in the promoters of housekeeping genes, and they might be expected to regulate the transcription of
various genes. In this study, we analyzed the GC-rich promoter of the
human gene for MAZ in an attempt to identify the role of GC-rich
cis-elements in the regulation of transcription of this gene.
The minimal MAZ promoter was located between nt
383 and +259 (Fig.
2A). We showed that a 135-base pair sequence, from nt
383
to
248 in the minimal promoter region of the gene for MAZ was
associated with the promoter activity. Further studies indicated that
the region from nt
383 to
334 was critical for the promoter activity (Fig. 2B). The G+C content of this region is
relatively low, and there are two CAAC elements and two CTTC elements
within this region (Fig. 2C). The region containing these
four elements is 33 base pairs long, with an average G+C content of
only 49%, the lowest G+C content in the extremely GC-rich promoter
region of the gene for MAZ. It has been reported that, in some
promoters, the proximal upstream region is extremely GC-rich, whereas
the distal region is AT-rich (46-49). It has also been reported that a
stretch of GC-rich sequences is followed by AT-rich sequences in some
promoters (49). A specific cis-element in the promoter region of the c-myc gene is localized in an AT-rich domain
that is flanked by GC-rich sequences (49). The cited studies suggest that relatively AT-rich elements in extremely GC-rich sequences might
be recognition sites for transcription factors that are associated with
the initiation of transcription (49). To determine whether these motifs
are critical for the activity of the promoter of the gene for MAZ, we
examined a series of constructs with mutations in these motifs. As
shown in Fig. 2D, two symmetric CAAC elements and two CTTC
symmetric elements were required for basal transcriptional activity,
and the contribution of each element to the total transcriptional activity was lower than that of all the elements together. Both CAAC
and CTTC elements have been found in the promoters of other genes, such
as the gene for the
-myosin heavy chain, the gene for
hydroxymethylbilane synthase, the gene for myosin light chain 2, and
the gene for lactoferrin, and some of these sites have been shown to be
important for promoter activity (42-45). The factors that bind to the
CAAC and/or CTTC elements in the minimal MAZ promoter remain to be identified.
The consensus sequence of MAZ-binding sites is very similar to that of
Sp1-binding sites. The GC-rich minimal promoter of the gene for MAZ
contains multiple binding sites for Sp1 and MAZ. We found that Sp1
bound to consensus Sp1-binding sites as well as to consensus
MAZ-binding sites. Similarly, MAZ bound to the consensus binding sites
for both MAZ and Sp1 (Fig. 3). The results of our gel shift assays
indicated that both Sp1 and MAZ recognized the same
cis-elements in the MAZ promoter. It has been reported that
a GC-rich motif in the c-myc promoter region is a high
affinity binding site for both MAZ and Sp1 (50). It has also been
reported that Sp1 binds to a series of GC-rich nucleotide sequences as well as to the consensus Sp1-binding site (51). The fact that MAZ and
Sp1 shared binding sites indicates that the regulatory activity of some
GC-rich cis-elements is consistent with cooperative interactions by multiple transcription factors, such as zinc finger proteins, with the same or overlapping cis-elements.
The binding of both Sp1 and MAZ to the same cis-elements in
the promoter region of the gene for MAZ might regulate transcription of
the gene. Both Sp1 and MAZ suppressed transcription from the MAZ
promoter (Fig. 4). There are seven consensus binding sites for Sp1 and
nine consensus binding sites for MAZ in the minimal promoter region of
the gene for MAZ. We tried to identify the cis-elements that
are involved in repression of the transcription of the gene for MAZ,
and we found that the extent of repression by Sp1 and by MAZ was
reduced only when all of the consensus binding sites for Sp1 and MAZ
had been mutated (pMAZCAT3-m8; Fig. 5). However, we did not detect
enhanced expression of the mutated construct (pMAZCAT3-m8), as compared
with that of the wild type construct, even when the possible consensus
GC-rich motifs in the promoter regions of the MAZ gene were mutated
(Fig. 5). We do not know the exact reason why the overexpression of Sp1
or MAZ stimulated the expression of pMAZCAT3-m8. One possible
explanation is that the weak binding sites of Sp1 or MAZ are still
present in pMAZCAT3-m8, which might be the other GC-rich sequences in the promoter region, and are functional for the residual activity of
repression. In fact, it has been reported that Sp1 or MAZ binds other
GC-rich elements besides the consensus motifs (27, 51). Further studies
are required for the identification of other motifs for Sp1 and MAZ.
Alternatively, we cannot rule out the possibility that the
overexpression of Sp1 and MAZ might titrate the coactivators or general
transcription factors and result in the repression of the MAZ promoter.
Further studies are required to answer these questions.
The activity of the promoter was repressed when any of the wild
type binding sites remained (pMAZCAT3-m1-pMAZCAT3-m7; Fig. 5),
indicating that almost all of the cis-elements were involved in repression. Autorepression of the gene for MAZ by MAZ itself also
indicates that negative feedback might possibly be involved in the
control of the expression of housekeeping genes. Both the multiple
GC-rich cis-elements and the upstream silencer element were
involved in the negative regulation of the gene for MAZ, indicating
that suppression of transcription of this gene is the major basal
regulatory mechanism that controls its expression.
Both Sp1 and MAZ repressed the activity of the gene for MAZ through
binding to the same cis-elements. We tried to determine whether the repression by Sp1 and by MAZ might be linked, but the
results failed to reveal any interaction between Sp1 and MAZ (data not
shown). Repression by Sp1 and repression by MAZ were independent
phenomena, even though both involved the same GC-rich cis-elements. We identified novel repressive domains in both
Sp1 and MAZ. The carboxyl-terminal region of Sp1 (amino acids 622-778) and amino acids 127-292 of MAZ were responsible for the respective repressive activities (Fig. 6, A and B).
Moreover, repression was also dependent on the zinc finger domains of
both Sp1 and MAZ, which were essential for binding to DNA (Fig. 6,
A and B). It is possible that Sp1 and MAZ might
bind to cis-elements through their zinc finger motifs,
recruiting other factors through their repression domains.
Histone deacetylases act negatively to regulate the expression of many
genes (52-54). Therefore, we examined whether histone deacetylases
might be involved in repression of the gene for MAZ. HeLa cells were
transfected with pCMV-Sp1 or pCMV-MAZ in the presence and absence of
TSA, a specific inhibitor of histone deacetylases. Only repression by
MAZ was released in the presence of TSA, whereas the repression by Sp1
was insensitive to treatment with TSA (Figs. 7A and
8A). Thus, it appears that histone deacetylases are involved in repression by MAZ. We confirmed this possibility by measuring HDAC
activity of immunoprecipitated complexes that contained MAZ. The HDAC
activity of complexes was about five times higher than that of control
immunoprecipitates, and the HDAC activity of the former complexes was
repressed in the presence of TSA (Fig. 7B). Immunoprecipitation and Western blotting analysis using nuclear extracts from HeLa cells indicated that MAZ could recruit proteins that
included HDAC1, HDAC2, and HDAC3 to form a multiple protein complex
(Fig. 7C).
We found that the action of Sp1 was insensitive to TSA and that HDAC1
had no effect on repression by Sp1 (Fig. 8A). Thus, repression mediated by Sp1 appeared to be independent of HDACs. It has
been reported that methylation plays an important role in the
suppression of transcription, and the interaction of Sp1 with MeCP2 has
also been reported (55). We examined whether methylation might be
involved in repression of the gene for MAZ and found that repression by
Sp1 was sensitive to 5-azacytidine, a specific inhibitor of methylation
(Fig. 8B). The ectopic expression of DNMT1 enhanced
repression by Sp1, whereas 5-azacytidine reversed the repression
induced by Sp1 and DNMT1 (Fig. 8C). Furthermore, it is
highly possible that DNMT1 is recruited by Sp1 (Fig. 8D). Therefore, DNMT1 appeared to play a role in the repression mediated by Sp1.
We have demonstrated here a possible mechanism for the down-regulation
and autorepression of a human housekeeping gene, namely the gene for
MAZ, through the recruitment of different repressors by two different
DNA-binding proteins, Sp1 and MAZ, which interact with the same
cis-elements (Fig. 9). Our
data suggest that different levels of suppression of the transcription
of this housekeeping gene might be responsible for the different levels
of expression of the gene in different tissues. Moreover, deacetylation
and methylation appear to play distinct roles in the regulation of a
single gene, namely the human gene for MAZ, in a process that is
mediated by different DNA-binding transcription factors.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 9.
Schematic representation of the regulation of
expression of the gene for MAZ by Sp1 and by MAZ. An Sp1-binding
site (Sp1-site) and a MAZ-binding site (MAZ-site)
are indicated.
|
|