In Drosophila SL2 cells, which lack the Sp family of tran-scription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter.. To understand the regulation
Trang 1Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteins
Shotaro Kishikawa1,2, Takehide Murata1, Hiromichi Kimura2, Kunio Shiota2and Kazunari K Yokoyama1 1
Gene Engineering Division, Department of Biological Systems, BioResource Center, RIKEN (The Institute of Physical & Chemical Research), Japan;2Department of Animal Resource Science/Veterinary Medical Sciences, The University of Tokyo, Japan
The Sp family is a family of transcription factors that
bind to cis-elements in the promoter regions of various
genes Regulation of transcription by Sp proteins is based
on interactions between a GC-rich binding site
(GGGCGG)in DNA and C-terminal zinc finger motifs
in the proteins In this study, we characterized the
GC-rich promoter of the gene for the DNA
methyl-transferase (Dnmt1)that is responsible for methylation of
cytosine residues in mammals and plays a role in gene
silencing We found that a cis-element (nucleotides )161
to )147)was essential for the expression of the mouse
gene for Dnmt1 DNA-binding assays indicated that
transcription factors Sp1 and Sp3 bound to the same
cis-element in this region in a dose-dependent manner In
Drosophila SL2 cells, which lack the Sp family of tran-scription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter Sti-mulation by Sp1 and Sp3 were independent phenomena Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3 The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3
Keywords: Dnmt1 gene; activation of transcription; Sp1; Sp3; p300
Transcription is regulated by the combinational actions of
proteins that bind to distinct promoter and enhancer
elements In general, a limited number of cis-acting
DNA elements is recognized, not that by a single
transcrip-tion factor exclusively but, rather, by a set of different
proteins that are structurally related [1] The promoter
regions of many eukaryotic genes contain GC-rich sequences
[2] and some of the most widely distributed promoter
elements are GC boxes and related motifs [2]
The Sp family of transcription factors includes the
proteins Sp1, Sp3 and Sp4, which recognize and bind to
GC boxes as well as to GT/A-rich motifs with similar
affinity, and Sp2, which binds preferentially to GT/A-rich
sequences [2,3] Sp1 and Sp3 are expressed in a wide variety
of mammalian cells whereas Sp4 has been detected
predominantly in neuronal tissues The regulation of gene
expression by Sp transcription factors is complex Although
certain promoters can be activated by either Sp1 or Sp3 in
assays in vivo and are occasionally activated by both Sp1
and Sp3 that act in a synergistic manner [4–6], there are
other promoters that show a definite preference for Sp1 or
Sp3 [7] Furthermore, Sp3 can function as an activator or a
repressor of transcription, depending on the gene in question [8,9]
The genes for several mammalian activators and repressors of transcription have been cloned The gene for p300 was first cloned as the gene for an E1A-associated protein with properties of a transcriptional adapter [10] The protein was found later to possess intrinsic histone acetyltransferase (HAT)activity and
to function as a coactivator in MyoD-, p53-, and SRC-1-mediated transcription [11,12] Furthermore, p300 appears to play a critical role in progression of the cell cycle and the differentiation of cells [11,12]
The methylation of DNA plays a role in the regulation
of gene expression [13,14], genomic imprinting [15] and inactivation of the X chromosome [16] and it has been shown to be essential for mammalian development [17,18] Altered patterns of DNA methylation has been implicated
in tumorigenesis [19] However, the mechanisms by which DNA methylation is regulated during development and tumorigenesis remain largely unknown Five distinct families of gene for DNA methyltransferases, designated Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L, have been identified in mammalian cells [20] Dnmt1 is expressed constitutively in proliferating cells, it is associated with foci
of DNA replication [21] and methylates CpG dinucleo-tides [22] These findings are consistent with the hypothesis that Dnmt1 is a maintenance methyltransferase that restores appropriate patterns of DNA methylation to the genome shortly after DNA replication [23] Representative sites for initiation of transcription have been found in the promoter of the Dnmt1 gene, namely, an oocyte-specific site, a somatic cell-specific site and a spermatocyte-specific site In adult somatic cells, most of the available data indicate that the identification by Bigey et al of many
Correspondence to K K Yokoyama, Gene Engineering Division,
Department of Biological Systems, BioResource Center, Tsukuba
Institute, RIKEN (The Institute of Physical & Chemical Research),
3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Fax: + 81 298 36 9120, Tel.: + 81 298 36 3612,
E-mail: kazu@rtc.riken.go.jp
Abbreviations: ChIP, chromatin immunoprecipitate; ODN,
oligodeoxynucleotides; HAT, histone acetyltransferase.
(Received 28 January 2002, revised 25 April 2002,
accepted 30 April 2002)
Trang 2sites of initiation of transcription of Dnmt1 [24] might
have been a mistake and that there is a single site only
[23]
To understand the regulation of expression of the
Dnmt1 gene in somatic cells, it is necessary to identify
and characterize the promoter region of this gene Previous studies showed that the 5¢ end region of the Dnmt1 gene is typical of a TATA-less and GC-rich promoter [24] However, specific cis- or trans-acting elements involved in the regulation of that promoter remain to be identified
In this study, we identified a cis-element located between nucleotides)161 and )147 that appeared to be activated independently by Sp1 and Sp3 Moreover, the p300 coactivator appeared to be involved in the Sp3-mediated activation of the mouse Dnmt1 promoter in somatic cells
M A T E R I A L S A N D M E T H O D S
Cells, plasmids and materials Mouse NIH3T3 cells and Drosophila SL2 cells were obtained from the JCRB Cell Bank (Tokyo, Japan)and from S Kojima at RIKEN (Tsukuba, Japan) pCMV-Sp1 and pGEX-Sp1 were provided by R Chiu of the UCLA School of Medicine (Los Angeles, CA, USA) Plasmids pPac, pPacSp1, pPacUSp3, pGEX-Sp3 and pCMV-Sp3 were gifts from G Suske at Philipps-Universita¨t (Mar-burg, Germany) GST–p300 (amino acids 1–596), GST– p300 (amino acids 744–1571)and GST–p300 (amino acids 1,572–2414)were obtained from Y Shi of Harvard Medical School (Boston, MA, USA) Plasmid pCi-p300 was a gift form Y Nakatani of the Dana Farber Cancer Research Institute (Boston, MA, USA) DNA fragments
of the mouse Dnmt1 promoter were excised with appro-priate restriction enzymes (D1 to D5 in Fig 1A) Each DNA fragment was inserted into the Nhe1 and Xho1 sites
of pGL3-basic (Promega Co., Madison, WI, USA) The integrating of all of the above recombinant plasmids was verified by sequence analysis Antibodies against Sp1 (PEP2), Sp3 (D-20), AP-2 (C-18)and p300 (C-20)were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA)
Fig 1 Characterization of the promoter of the mouse Dnmt1 gene (A) Transcriptional activity of the mouse Dnmt1 promoter in NIH3T3 cells The top diagram shows the mouse Dnmt1 gene and the lower diagram shows the variously deleted promoters fused to a gene for luciferase (gray rectangle) The corresponding luciferase activities are also shown Numbering is relative to the first site of initiation of transcription (+1) Reporter plasmid D5 has the deletion of nucleo-tides )173 to )120 of D2 fusion construct NIH3T3 cells were trans-fected with plasmids that encoded the various constructs and luciferase activity was measured as described in the text Promoter activities are expressed relative to the activity associated with reporter plasmid D4, which was taken arbitrarily as 1.0 All values are the averages of results from at least three experiments and the standard deviation for each value is indicated (B)Nucleotide sequence of the 5¢ flanking region of the mouse gene for Dnmt1 Bases are numbered relative to the site
of initiation of transcription (+1) P1, P2 and P3 indicate the probes used for EMSAs (C)Binding of transcription factors to the Dnmt1 promoter Binding of transcription factors to the region between nucleotides )173 and )117 of the Dnmt1 promoter was analyzed by EMSAs with 32 P-labeled probes P1, P2, and P3 (1 · 10 4 c.p.m)and
5 lg protein of nuclear extract (NE)from NIH3T3 cells Three shifted protein-DNA complexes are indicated by arrows (B1–B3).
Trang 3Site-directed mutagenesis
Site-directed mutagenesis was performed with a
Quick-Change Site-Directed Mutagenesis Kit (Stratagene,
La Jolla, CA, USA)using various oligodeoxynucleotide
primers (n1 to n10 in Fig 2D)and a fragment of the
Dnmt1 promoter (nucleotides)220 to +79)as template
The mutated DNA fragments were subcloned into the
Nhe1 and Xho1 sites of pGL3-basic (Promega Co.) The
integrity of all of the vectors was verified by sequence
analysis
Recombinant proteins
Glutathione S-transferase (GST), GST–Sp1, GST–Sp3,
GST–p300 (amino acids 1–596), GST–p300 (amino acids
744–1571)and p300 (amino acids 1572–2414)were prepared
as described previously [25].35S-Labeled Sp1 and Sp3 were
synthesized in a TNT Reticulocyte Lysate System (Promega
Co.), according to the manufacturer’s protocol
Cell culture, transfections and assays of promoter
activity
Mouse NIH3T3 and F9 cells and human HeLa and 293
cells were grown in Dulbecco’s modified Eagle’s medium
(Nissui Pharmaceutical Co., Tokyo, Japan)with 10% fetal
bovine serum (Invitrogen BV, Groningen, the Netherlands)
at 37C in a humidified atmosphere of 5% CO2 in air
Schneider’s Drosophila SL2 cells were maintained in Shields
and Sang M3 insect medium (Sigma–Aldrich Japan Co.,
Tokyo, Japan)supplemented with 10% fetal bovine serum
at 22C in air One day prior to transfection, mammalian
cells were seeded in 12-well plates at 8· 104cells per well
and SL2 cells were seeded at 1· 106cells per well Cells
were transfected with the indicated amount of reporter
plasmid using LipofectamineTM2000 (Invitrogen BV) Cell
extracts were prepared 24 or 48 h after transfection
Promoter activity was determined with a Luciferase Assay
System (Promega Co.)as described by the manufacturer
Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts were prepared from NIH3T3, HeLa, 293
and F9 cells and EMSAs were performed as described
previously [26] DNA probes were radiolabeled with T4 polynucleotide kinase (New England BioLabs Inc., Beverly,
MA, USA)and [c-32P]ATP (Amersham Pharmacia
Fig 2 Identification of a cis-element in the Dnmt1 promoter (A)
EMSAs were performed with mutated oligodeoxynucleotides as
competitors and the wild-type ODN probe (B)Oligodeoxynucleotides
used as competitors are listed Wt, Sequence of the wild-type probes.
M1 through M11, the GAATTC sequence was introduced into the
wild-type motif to generate the mutant sequences (C)Assays of
luciferase reporter activity in NIH3T3 cells using mutated reporter
constructs NIH3T3 cells were transfected with the reporter plasmid
D2 or with plasmids that included the sequences n1 through n10 and
luciferase activities were measured, and compared with that obtained
with NIH3T3 cells that had been transfected with reporter plasmid D4
(500 ng) These assays were repeated at least three times and the
standard deviation for each average value is indicated (D)Nucleotide
sequences obtained by site-directed mutagenesis Wt, wild-type
sequence; n1 through n10, TT di-nucleotides were introduced, as
indicated, to generate the mutants.
Trang 4Biotech., Uppsala, Sweden) The binding reaction was
performed in 20 lL of buffer that contained 25 mM
N-2-Hydroxyethylpiperazine-N¢-2-ethanesulfonic acid/KOH
(Hepes/KOH, pH 7.9), 25 mMKCl, 5 mMMgCl2, 50 mM
ZnSO4, 1 lg poly(dI-dC), and a nuclear extract or purified
GST-fusion protein In some cases, competitors or
anti-bodies were added to reaction mixtures which were then
incubated on ice for 20 min After addition of the
32P-labeled DNA probe, the mixture was incubated for a
further 20 min on ice Products of reactions were resolved
on a 5% polyacrylamide gel in 0.5· Tris/borate/EDTA
buffer Electrophoresis was performed at 180 V for 3 h at
4C
Immunoprecipitation of chromatin (ChIP)
NIH3T3 cells were fixed in 1% formaldehyde at room
temperature for 15 min Chromatin was prepared with a
kit from Upstate Biotechnology (Lake Placid, NY, USA)
according to the recommendations of the manufacturer,
with eight 10-s pulses of sonication at 10-s intervals, which
yielded chromatin fragments of 1.0-kb in length
Equivalent amounts of chromatin were
immunoprecipi-tated with the indicated antibody or with irrelevant IgG,
as a control, at 4C for 5 h Immunocomplexes were then
recovered by addition of protein A/G PLUS-agarose
beads (Santa Cruz Biotech., Santa Cruz, CA, USA)with
incubation at 4C for 2 h After the beads had been
washed extensively, DNA was eluted and cross-linking
was reversed by addition of 200 lL of elution buffer (1%
SDS/0.1M NaHCO3)and incubated overnight at 65C
DNA was extracted with phenol/chloroform (1 : 1, v/v),
precipitated in ethanol and then analyzed by PCR with
primers that corresponded to the cis-element (nucleotides
)220 to +79), namely, 5¢-AAGGCTAGCCAGAGTCA
TCCTCTGC-3¢ (forward direction)and 5¢-GCGCTCG
AGCTTGCAGGTTGCAGAC-3¢ (reverse direction) PCR
was performed for 35 cycles and products were analyzed
by agarose gel electrophoresis
Immunoprecipitation and Western blotting
Cell pellets were lysed in RIPA buffer (1· NaCl/Pi,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 100 lgÆmL)1phenylmethanesulfonyl fluoride, 1 mM
sodium orthovanadate and 2 lgÆmL)1aprotinin]
Whole-cell extracts (500 lg proteins)were incubated with 2 lg of
antibody [Sp1 Ig, Sp3 Ig, (AP-2)Ig,
anti-p300 Ig or nonimmunized rabbit IgG] for 1 h, and then
30 lL of a suspension of protein A/G PLUS-agarose
beads (Santa Cruz)were added After incubation for 1 h
at 4C, immunoprecipitates were gently washed three
times with NaCl/Pi, boiled and subjected to SDS/PAGE
(7% acrylamide gel) Proteins were electroblotted onto a
poly(vinylidene difluoride)membrane filter and blocked
for 1 h in Blotto A [10 mM Tris/HCl (pH 8.0), 150 mM
NaCl, 5% skim milk, 0.05% Tween-20], and then
incubated with 2 lgÆmL)1of primary antibody (anti-Sp1
Ig or anti-Sp3 Ig)in BlottoA for 1 h Finally, 30 lL of a
solution of horseradish peroxidase conjugated-secondary
antibody (New England Biolabs)in Blotto A were added
Antibody–HRP complexes were detected with ECL
West-ern blotting detection reagents (Amersham Pharmacia
Biotech.)according to the instructions from the manufac-turer
‘GST-pull down’ assay Two micrograms of GST–protein and 35S-labeled Sp3 (3· 103c.p.m)that had been in vitro translated in a final volume of 500 lL of binding buffer [250 mMNaCl, 50 mM Hepes/KOH (pH 7.5), 0.5 mMEDTA, 0.1% Nonidet P-40, 0.2 mMphenylmethanesulfonyl fluoride, 1 mM dithiothre-itol and 500 lgÆmL)1BSA], were incubated at 4C for 1 h and then 30 lL of a suspension of glutathione–Sepharose 4B (Amersham Pharmacia Biotech)were added After incubation for 1 h at 4C, samples were gently washed three times with NaCl/Pi, boiled and fractionated by SDS/ PAGE (7% acrylamide gel)
R E S U L T S
Identification ofcis-elements that control expression of the Dnmt1 gene
We subcloned a 2.0-kb DNA fragment that contained the Dnmt1 promoter region from a mouse genomic clone into pGL3-basic, a vector that includes a gene for luciferase without any eukaryotic promoter or enhancer elements We then generated a series of 5¢-serial-deletion constructs of the promoter–luciferase gene for transfections and subsequent assays of luciferase activity (Fig 1A) Constructs D1 to D5, together with the pGL3-basic vector, were used for transient transfection of NIH3T3 cells in an attempt to identify the cis-elements of the gene for Dnmt1 and to delineate the 5¢ boundary of the promoter As shown in Fig 1A, weak control of promoter activity was associated with a region of
1827 bp in the upstream region between nucleotides)2000 and)173, with no more than 40% variation in activity A large reduction of approximately sixfold in promoter activity was detected when we removed nucleotides)173
to )120 (Fig 1A, D3 to D5), suggesting that cis-acting elements that are critical for Dnmt1 promoter activity might
be located in this region We detected a single site for initiation of the transcription of Dnmt1 at +1 in this promoter (data not shown), a result that is consistent with previous reports [20,23] Thus, it appeared that the region from nucleotides )173 to )120 contained critical cis-elements for transcriptional activation of the Dnmt1 gene
To examine DNA-binding proteins, we prepared DNA probes from the promoter of the mouse gene for Dnmt1 (nucleotides )173 to )117), which contained putative binding sites for Sp1, and performed gel shift assays as described previously [26] Three DNA probes, P1 (nucleo-tides)173 to )138), P3 (nucleotides )160 to )131)and P2 (nucleotides )141 to )117)were prepared (Fig 1B) EMSAs of nuclear extracts from NIH3T3 cells with the P1 probe revealed shifts of three bands on the gel (B1 to B3
in Fig 1C) The intensity of these bands was significantly enhanced with P3 probe and no DNA–protein complexes were evident when we analyzed nuclear extracts of NIH3T3 cells with the P2 probe We observed three similar shifted bands when we used nuclear extracts from 3T6 cells, HeLa cells, 293 cells and F9 cells (data not shown) To define the binding site in this region more precisely, we synthesized 11 mutant oligodeoxynucleotides (ODNs; M1 to M11 in
Trang 5Fig 2B)and used them as competitiors in EMSAs
(Fig 2A) The DNA–protein complexes B1, B2, and B3
were detected with the wild-type P1 probe After addition of
a 100-fold excess of unlabeled mutant ODN (M1 to M11)to
the reaction mixture, we found that shifted bands were not
eliminated by the unlabeled mutant ODNs M5 through M8
The nucleotide sequences of these ODNs were
com-pared and the consensus-binding site was defined as the
15-bp sequence 5¢-GGCAAGGGGGAGGTG-3¢ (Fig 2B),
which we designated the GA motif To examine whether or
not this sequence is critical for the transcriptional activity of
the Dnmt1 promoter, we synthesized 10 mutant ODNs
(n1 to n10 in Fig 2D)and introduced them into luciferase
constructs to generate the respective reporter plasmids We
examined the activity of these luciferase reporters in
NIH3T3 cells and found that only the n5 construct lacked
transcriptional activity (Fig 2C) Thus, the central GG
dinucleotide in the 15-bp motif seemed to be critical for the
transcriptional activity of the Dnmt1 promoter
Sp1 and Sp3 bind to the GA motif in the Dnmt1 promoter
To identify the transcription factors that bind to the GA
motif in the Dnmt1 promoter, we looked for transcription
factors in the TRANSFAC database [27] We failed to
identify known factors that bind to this sequence However,
we found that both AP-2 and Sp1 bound to sequences that
exhibited strong similarity, namely 0.859 and 0.856,
respectively, to the GA motif To determine whether Sp1
and/or AP-2 could bind to the GA consensus sequence, we
performed EMSAs in the presence of antibodies against
Sp1, Sp3 and AP-2 (Fig 3A) The retarded band designed
B1 was shifted even further upon addition of antibodies
against Sp1 (lane 3), while the retarded bands corresponding
to B2 and B3 were shifted further upon addition of
antibodies against Sp3 (lane 4) Both antibodies against Sp1
and against Sp3 affected the migration of all retarded bands
In contrast, anti-(AP-2)Ig did not affect the migration of
shifted bands (lane 6) Thus, it appeared that both Sp1
and Sp3 bound to the 5¢-GGCAAGGGGGAGGTG-3¢
sequence in the Dnmt1 promoter in vitro We also performed
a chromatin immunoprecipitation experiment to assess the
interaction of Sp1 and Sp3 in the transcription of the Dnmt1
gene at the chromatin level (Fig 3B) In contrast to the
absence of any effect of IgG, immunoprecipitates obtained
with antibodies both against Sp1 and against Sp3 yielded a
318-bp DNA fragment after PCR that included the 15-bp
sequence Antibodies against AP-2 did not generate the
318-bp DNA (data not shown) These results supported our
hypothesis that both Sp1 and Sp3 bind to cis-elements in the
gene for Dnmt1 that include the GA motif
Stimulation of transcription from the Dnmt1
promoter by Sp1 and Sp3 in SL2 cells
Both Sp1 and Sp3 bound to the Dnmt1 promoter
Therefore, we next examined the effects of Sp1 and Sp3
on the expression of the gene for Dnmt1 We transfected
DrosophilaSL2 cells with a luciferase reporter construct in
the presence and absence of the expression plasmids
pPacSp1 and pPacUSp3, respectively SL2 cells lack
endogenous Sp proteins, and, thus, the
cis-element-depend-ent activation of the Dnmt1 promoter is dependcis-element-depend-ent on the
gene products of exogenously introduced genes that encode Sp1 or Sp3 The activity associated with the luciferase reporter construct D2 was stimulated in the presence of pPacSp1 (Fig 4A)and in the presence of pPacUSp3
Fig 3 Sp1 and Sp3 bound to a cis-element in the promoter of the mouse gene for Dnmt1 (A)32P-Labeled probe P3 (1 · 10 4 c.p.m)was incu-bated with 5 lg protein of nuclear extract (NE)from NIH3T3 cells in the presence and absence of antibodies against Sp1, Sp3 or AP-2 Lane
3, Sp1-specific antibody (2 lg); lane 4, Sp3-specific antibody (2 lg); lane 5, Sp1-specific and Sp3-specific antibodies (2 lg each); lane 6, AP-2-specific antibody (2 lg) (B) Chromatin immunoprecipitation assays Chromatin immunoprecipitation assays were performed as described in the text DNA and proteins were cross-linked with formaldehyde, and DNA was sheared and immunoprecipitated with Sp1- or Sp3-specific antibody (2 lgÆmL)1each) After reversal of cross-links, DNA was amplified with primers specific for the promoter region of the Dnmt1 gene Products of PCR were resolved by agarose gel electrophoresis The arrowhead indicates the amplified DNA fragment (318 bp).
Trang 6(Fig 4B) Moreover, the promoter activity of D2 was
further enhanced in the presence of both pPacSp1 and
pPacUSp3 (Fig 4E) In contrast, the activity associated
with the luciferase reporter construct n5 was not stimulated
by either pPacSp1 or pPacUSp3 (Fig 4C,D) These results
indicate that both Sp1 and Sp3 enhanced transcription from
the Dnmt1 promoter
Independent activation of the Dnmt1 promoter
by Sp1 and Sp3 through the same GA motif
Sp1 and Sp3 bound to the same cis-element in the promoter
of the gene for Dnmt1 Therefore, we next examined
whether activation by Sp1 or by Sp3 affect expression of the
gene for Dnmt1 In an attempt to identify whether Sp1 and
Sp3 could bind to the same GA motif, we prepared
appropriate GST fusion proteins and performed EMSAs
with the P3 probe that included the GA motif Specific and
different shifted bands were detected with GST–Sp1 and
GST–Sp3 The shifted band due to GST–Sp3 gradually
disappeared when increasing amounts of GST–Sp1 were
added as competitor (Fig 5A,B) In contrast, the intensity
of the shifted band that included GST–Sp1 gradually
increased Conversely, the addition of GST–Sp3 resulted in
a similar reduction in the binding of Sp1 to the GA motif
(data not shown) These results imply that the binding of
Sp1 and of Sp3 to the GA motif were independent
phenomena and that each competed for binding to DNA with the other through the same cis-element
We next examined whether Sp1 might be included in transcriptional complexes with Sp3 in vivo Western blotting analysis with antibodies against Sp1 of immunoprecipitates obtained with antibodies against Sp3 indicated that Sp1 was not immunoprecipitated with Sp3 from extracts of NIH3T3 cells (Fig 5C) Complementary studies of immunoprecipi-tates obtained with Sp1-specific antibodies and Western blotting with antibodies against Sp3 also indicated that Sp1 and Sp3 did not interact with each other (data not shown)
We also examined the molecular association of GST–Sp1 and GST–Sp3 fusion proteins and found no evidence of any association in vitro (data not shown)
Enhancement by p300 of transcription from the Dnmt1 promoter that is induced by Sp3
The transcriptional coactivator p300 mediates growth arrest
by catalyzing histone acetylation and the subsequent rearrangement of chromatin [11,12] Recent reports indicate that p300 also collaborates with Sp1 or Sp3 to regulate the expression of the promoter of the gene for p21Waf1/Cip1 [28,29] Therefore, we examined the effect of p300 on the promoter activity of the Dnmt1 gene in the presence of pCMV-Sp1 and of pCMV-Sp3 in NIH3T3 cells As shown
in Fig 6, cotransfection with pCi-p300 and pCMV-Sp3
Fig 4 Activation of transcription from the Dnmt1 promoter by Sp1 and Sp3 in Drosophila SL2 cells SL2 cells were transfected with 500 ng of the Dnmt1 reporter plasmid D2 (A,B,E)and the reporter plamid n5 without GA motif (C,D)and the indicated amounts of pPacSp1 (A,C), pPacUSp3 (B,D)or both pPacSp1 and pPacUSp3 (E)(each 500 ng)and then luciferase activities were measured The total amount of the plasmid DNA (pPacSp1 or pPacUSp3)was adjusted to 1 lg with pPac (no insert) Assays were repeated at least three times and the standard deviation for each mean value is indicated.
Trang 7enhanced the reporter activity controlled by the Dnmt1
promoter, but cotransfection of pCi-p300 with pCMV-Sp1
did not The extent of activation was much higher than that
obtained with pCi-p300 and with pCMV-Sp3, indicating
that p300 enhanced the promoter activity of the Dnmt1 gene
that was induced by Sp3, but not by Sp1 Further studies of
transactivation using a GAL4 fusion with p300 and the
dominant negative form of p300 are required for a full
understanding of the molecular mechanism of this
phe-nomenon
Sp3 interacts with the C-terminal region of p300
To determine whether Sp3 associates directly with p300,
we performed immunoprecipitation and Western blotting
assays using antibodies against p300 and Sp1 or Sp3 and extracts of NIH3T3 cells We immunoblotted immunopre-cipitates obtained with p300-specific antibodies with anti-bodies against Sp1 or against Sp3 As shown in Fig 7A, a p300-specific band was detected with antibodies against Sp3 but not against Sp1 Antibodies specific for AP-2 (data not shown)and control IgG did not yield any evidence of interactions with Sp3 To determine which regions of p300 interacted with Sp3, we prepared 35S-labeled Sp3 by translation in vitro and investigated the binding to various GST–p300 fusion proteins by the GST-pull down assay [25] Only the C-terminal region of p300 (amino acids 1572–2414), which included the C/H region and E1A-binding region of p300, was found to associate with Sp3 (Fig 7B) We performed similar complementary experi-ments to determine which parts of p300 interacted with Sp3 and found that the [35S]Met-labeled carboxyl region of p300 interacted with GST–Sp3 (data not shown) These results indicated that Sp3 was able to bind to the C-terminal domain of p300
D I S C U S S I O N
The promoters of many housekeeping genes have a number of common characteristics, such as the presence
of multiple sites for initiation of transcription which, presumably, compensate for the absence of a TATA box and a CAAT box, and they often have an unusual high GC-content [1] The Dnmt1 gene is also a housekeeping
Fig 5 Binding of Sp1 and Sp3 to the cis-element in the promoter of the
Dnmt1 gene (A)EMSAs of GST-Sp1 and GST-Sp3 fusion proteins
with the P3 probe Indicated amounts of GST-Sp1 (onefold to 50-fold
excess)were added to a reaction mixture that included 0.1 lg of
GST-Sp3 and the P3 probe (0.2 nmolÆmL)1) (B) Relative binding of Sp1
and Sp3 to P3 in EMSA The results in (A)are summarized as relative
DNA-binding activity (%) The total intensity of P3 probe on a
reaction mixture for EMSAs was taken arbitrarily as 100% (C)Sp1
and Sp3 do not form a stable complex in NIH3T3 cells
Immuno-blotting of Sp1 in the immunoprecipitates derived from 500 lg protein
of whole-cell extracts of NIH3T3 cells with Sp3-specific antibody
(2 lgÆmL)1) Sp1 did not form a complex with Sp3 in NIH3T3 cells.
Input: this lane was loaded 50 lg protein of whole-cell extracts.
Fig 6 Enhancement of the Dnmt1 promoter activity upon cotransfec-tion with p300- and Sp3-expression plasmids NIH3T3 cells were transfected with 0.5 lg of the Dnmt1 gene reporter plasmid D2 (see Fig 1A)plus 0.5 lg of pCMV-Sp1, pCMV-Sp3 or/and pCi-p300 and luciferase activity was measured in each case Total amounts of DNA (pCMV-Sp1, pCMV-Sp3 and pCi-p300)were adjusted to 2 lg with pBSII KS(+) The assay was repeated at least three times and the standard deviation for each value is indicated.
Trang 8gene [24] However, details of the cis-elements in its
promoter and the factors that regulate expression of the
Dnmt1 gene from the minimum promoter in somatic cells
remain to be determined In this report, we identified a
cis-element in the promoter of the Dnmt1 gene in somatic
cells and showed that both Sp1 and Sp3 bound to this
regulatory element, the GA motif, independently
More-over, both Sp1 and Sp3 stimulated the promoter activity
of the Dnmt1 gene and Sp3 was found to associate with
p300 through the C-terminal region of the latter protein
and to enhance its activity
In order to identify the minimal promoter in a 2.0-kb
region of the Dnmt1 gene, we generated a series of
promoter–luciferase deletion constructs for reporter assays and then determined the luciferase activity associated with each respective construct (Fig 1A) The results indicated that a cis-acting region of 53-bp was located between nucleotides)173 and )120 Precise dissection of the region
by EMSAs demonstrated that a minimal element, from nucleotides)161 to )147, was critical for the activation of transcription of the Dnmt1 gene (Fig 2A) We identified a
GA motif in this region required for the binding of SP1 and SP3 to the DNA (Fig 3A,B)in order to activate transcrip-tion of the Dnmt1 gene (Fig 4) EMSAs in the presence of anti-Sp1 Ig and anti-Sp3 Ig confirmed these results (Fig 3A) The GA motif is different from the typical sequence of Sp1-binding sites, GGGCGG (Fig 2) Thus, Sp1- and Sp3-binding sites might be affected by sequences adjacent to GC-rich or GA-rich elements that influence maximum binding It has been reported that Sp1 and Sp3 might be involved in the activation of a very large number of genes, such as housekeeping genes for tissue-specific and cell cycle-regulated proteins [3] Moreover, Sp proteins are involved not only in activation but also in repression We studied the effects on the expression of the Dnmt1 gene by Sp1 or Sp3, which bound to the similar elements (Fig 3) The two proteins are found in the same cells and are indistinguishable in terms of DNA-binding specificity (Fig 5A,B), and both proteins bound independently to the GA motif (Fig 5C) All Sp proteins contain three zinc fingers close to the C-terminus, with glutamine-rich domains adjacent to serine/threonine structures in the N-terminal region Sp1, Sp3 and Sp4 are more closely related to each other than they are to Sp2 [3] The homology among the zinc fingers of all known Sp proteins is close to 90%, but, the homology among entire sequences is close to 40%
In the transcriptional activation of the Dnmt1 gene, both Sp1 and Sp3 play a critical role (Fig 4)via binding to the same cis-element (GA motif)and the binding of each is independent of the other (Fig 5) A competition experiment
in vitrowith the GA motif demonstrated that each factor is able to replace the other in terms of binding to DNA The growth characteristics of Sp1-deficient embryonic stem cells (ES cells)are normal and such cells can be induced
to differentiate [30] Nevertheless, Sp1 is essential for normal mouse embryogenesis and the development of Sp1-knock-out embryos is severely retarded, with death occurring around day 11 of gestation Thus, Sp1 appears to be a transcription factor whose function is essential after day 10
of development Other Sp proteins, such as Sp3, might be able to compensate, at least in part, for the loss of Sp1 activity at early embryonic stages Sp3 is expressed ubiqui-tously and has the potential to activate transcription Moreover, its DNA-binding activity is indistinguishable from that of Sp1 A recent study of Sp3 null-mice suggested that Sp1 and Sp3 might have similar and therefore redundant functions during early development but might have distinct and highly specific functions at later stages of development [31] Thus, Sp1 and Sp3 might have a wide range of redundant functions and might be able to replace each other in Sp1-and Sp3-knockout mice [31] It was reported that three isoforms of Sp3 exist and that these different isoforms play different roles in the activation and repression of transcription [9] In our hands, Sp1 generated
a single band and Sp3 generated two bands in EMSAs (two
of three bands migrated to the same position; B3 in
Fig 7 p300 was associated with Sp3 (A)p300 associated with Sp3 and
not with Sp1 in NIH3T3 cells p300 was immunoprecipitated with
anti-p300 Ig (2 lg)from 500 lg protein of whole-cell extract of NIH3T3
cells and the immunoprecipitate was immunoblotted with antibodies
against Sp3 (2 lgÆmL)1; upper panel)or Sp1 (2 lgÆmL)1; lower panel)
as described in the text (B)Direct interaction of Sp3 with various
GST–p300 deletion mutants 35 S-Labeled Sp3 (3 · 10 3 c.p.m)was
incubated with 2.0 lg of each GST–p300 mutant, as indicated (lanes
3–5), or with 2.0 lg GST alone (lane 2) Lane 1, one tenth of input
35 S-labeled Sp3 After electrophoresis, radiorabeled protein was
detected by autoradiography The shaded boxes of p300 indicate the
C/H1 domain, C/H2 domain and C/H3 domain [10].
Trang 9Fig 3A) It has been suggested that two small isoforms of
Sp3 might act as repressor molecules with full-length Sp3
acting as an activator [3,9] Sp3 acts as a transcriptional
activator at many promoters, as does Sp1 [5,32] In studies
of other promoters such as the uteroglobin gene [8],
monocyte chemoattractant protein-1 gene and ornithine
decarboxylase gene [7,33], however, Sp3 was found to be
inactive or to act as only a weak activator or as a repressor
of Sp1-mediated transcription
The most obvious differences between Sp1 and Sp3 are
the presence of a potent inhibitory domain in Sp3 [34] The
relative abundance of Sp1 and Sp3 allows the fine tuning of
the regulation of gene activities In endothelial cells that
contain high levels of both Sp1 and Sp3, the ratio of Sp1 to
Sp3 is higher than in nonendothelial cells [35] In primary
keratinocytes, levels of Sp3 exceed those of Sp1 The ratio of
Sp3 to Sp1 is inverted when these cells differentiate in vitro
In differentiating keratinocytes, only Sp3 enhances the
activation of the promoter of the gene for p21Waf1/Cip1[36]
A change in the ratio of Sp1 to Sp3 also occurs when C2C12
myocytes are cultivated under hypoxic conditions Hypoxia
causes the progressive depletion of Sp3, whereas the level of
Sp1 remains unchanged [37] It has been demonstrated that
the expression of the gene for Dnmt1 is regulated in a
cell-cycle-dependent manner The expression of Dnmt1 is
enhanced during the S phase and then declines with the
approach of the M phase In a preliminary study, we found
that levels of expression of Sp1 and Sp3 differed during the
cell cycle; Sp1 was expressed predominantly at the G1 phase
and Sp3 was expressed at S phase (data not shown)
Therefore, it is quite plausible that Sp1 and Sp3 might
control the expression of the Dnmt1 gene Further studies
are required to clarify the distinct roles of Sp1 and Sp3 at
different phases of the cell cycle
The transcriptional cofactor p300 is coprecipitated in
complexes with Sp1 [38] The activation of the promoter of
the gene for p21Waf1/Cip1 by butyrate and nerve growth
factor requires functional collaboration between Sp1 and
Sp3 [28,38] However, although p300 and Sp1 are
compo-nents of the complex that activates the promoter of the gene
for p21Waf1/Cip1, the interaction is indirect Thus, p300 is
required for the tricostatin A-induced (TSA-induced),
Sp1-mediated transcription of the gene, but details of the
interaction between Sp1 and p300 in this phenomenon are
unknown It is possible that Sp3 might bind to a GC or GA
motif in the promoter of the gene for p21Waf1/Cip1through
binding with p300, as such motifs might bind Sp1 and Sp3
Thus, Sp3 might regulate the TSA-dependent activity of the
promoter We showed that Sp3 might associate with the
C-terminal region of p300 by direct binding (Fig 7B) This
region is similar to the region to which GATA-1, E2F and
p53 also bind [10] Thus, regulation by Sp1 and Sp3 of target
genes might be involved in the cell cycle Furthermore, the
activity of p300 is dependent on gene dosage during early
embryogenesis Thus, it is possible that Sp3 might compete
for binding to DNA with Sp1-like proteins and
Kru¨ppel-like factors via interactions with p300 at different phases of
the cell cycle in somatic cells In conclusion, we propose that
the nucleotide sequences to which Sp1 and Sp3 bind are
similar and binding of these factors is independently
regulated by different coactivators in a cell-cycle-dependent
manner The distinct functions of Sp1 and Sp3 in the
regulation of expression of the Dnmt1 gene during the cell cycle remain to be clarified
A C K N O W L E D G E M E N T S The authors thank Drs G Suske, R Chiu, Y Shi, Y Nakatani,
S Kojima, H Ugai, C Jin, J Song and A Wolff for plasmids, reagents and valuable discussions This work was supported by the Special Coordination Funds of RIKEN, by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan (to
K K Y)and a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (to K S).
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