From the Dipartimento di Biochimica e Biotecnologie
Mediche, Università degli Studi di Napoli `Federico II', Via S. Pansini 5, I-80131 Napoli, Italy and the
Dipartimento di
Medicina Sperimentale e Clinica, Università degli Studi di
Catanzaro, Via T. Campanella, I-88100 Catanzaro, Italy
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ABSTRACT |
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We report that the heterotrimeric transcription
factor NFY or "CAAT-binding factor" binds the Transcription of the human H ferritin gene is driven by two
promoter elements, A and B, located 170 base pairs upstream from the
transcription start site (1). Element A of the ferritin promoter,
located NFY is a ubiquitous transcription factor formed by subunits A, B, and C
(6). NFY-B and NFY-C subunits contain a conserved histone-fold motif,
which forms a dimer ("histone-fold") and interacts with subunit A
(7). NFY specifically recognizes a CAAT-box motif found in the promoter
and enhancer regions of many eukaryotic genes (8). Among the genes
recognized by NFY, some are induced by cAMP. The promoter region
responsible for the cAMP-mediated stimulation of transcription includes
the CAAT element recognized by NFY (4, 9, 10).
We report that NFY is a component of the Bbf complex. Also, we
demonstrate that NFY interacts, both in vivo and in
vitro, with the co-activator molecule p300/CBP. Using
antibodies against the three NFY subunits and p300 protein segments, we
found that NFY-B subunit binds the central region of p300 (residues
671-1194).
Cell Culture--
HeLa cells were cultured as monolayers in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.),
supplemented with 10% (v/v) fetal calf serum (Sigma), 100 units/liter
penicillin (Hyclone Labs.). Cells were grown at 37 °C in a 5%
CO2 atmosphere.
Plasmids--
The expression vectors for p300 were kindly
donated by Dr. V. Ogryzko (NIH, Bethesda, MD). For the expression in
eukaryotic cells, different regions of p300 were cloned in the pCI
vector (Promega) under the control of the cytomegalovirus promoter.
p300 N encodes amino acids 1-670; p300 M encodes amino acids
671-1194; and p300 C encodes amino acids 1135-2414. All these
proteins are flag-tagged. For the expression in prokaryotic cells (BL21
DE3 strain), the N, M, and C regions of p300 were cloned in pet28c vector (Novagen). All these proteins are flag-tagged. The Bbf-TATAA construct is described elsewhere (4); it contains a B-site oligonucleotide (5'-CGGCGCTGATTGGCCGGGGCGGGC-3') fused to
the ferritin TATAA box in the pEMBL8-CAT vector.
DNA Transfections, Cell Extracts, and CAT Assays--
HeLa cells
were transfected with 5 µg of DNA using the calcium phosphate
co-precipitation method (11). Cells washed twice in cold
phosphate-buffered saline and collected by centrifugation were used for
immunoprecipitation and Western blot analysis. The pellet was
resuspended in 500 µl of lysis buffer containing 250 mM
NaCl, 20 mM NaH2PO4, pH 7, 30 mM sodium pyrophosphate, 5 mM EDTA, 1 mM NaF supplemented with 5 mM dithiothreitol,
10 µg/ml of leupeptin, 10 µg/ml of pepstatin, 10 µg/ml aprotinin,
0.1 mM Na3O4V, 1 mM
phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40. After incubation
for 10 min on ice, the suspension was centrifuged at 14,000 × g for 10 min.
Five micrograms of a plasmid carrying the luciferase gene controlled by
the cytomegalovirus enhancer were included in the transfection
experiments followed by CAT assay. The cell extracts and the CAT and
luciferase assays are described elsewhere (4).
Immunoprecipitations--
HeLa cell extract (1 mg) was incubated
with 50 µl of anti-flag M2 affinity gel for 1 h at 4 °C. The
beads were collected by centrifugation, washed four times with the
lysis buffer, and then loaded on a 12% SDS-PAGE gel.
Western Blot Analysis--
After electrophoresis, the proteins
were transferred to nitro-cellulose. Then the blocking mixture was
added, and the membrane was incubated with a 1:5000 dilution of rabbit
anti-NF-YA, NF-YB, or NF-YC antibodies or with a 1:1000 dilution of
mouse anti-flag or mouse anti-RII Expression of Recombinant Proteins--
p300 N, p300 M, and p300
C-pet 28 plasmids were expressed and purified from soluble fraction of
Escherichia coli BL21 (DE3) after induction with 0.4 mM isopropyl- Nuclear Extracts and EMSA--
Nuclear extracts were prepared
from HeLa cells as described previously (4). The B-site oligonucleotide
used for EMSA was 5'-CGGCGCTGATTGGCCGGGGCGGGC-3'. To study DNA-protein
interaction on the B site, we also used the region The Trimeric Transcription Factor NFY Binds H Ferritin
Promoter--
The
We next investigated the effect of NFY specific antibodies on the
natural promoter fragment (
These data demonstrate that NFY binds the B box of the ferritin
promoter and suggest that, in vivo, other components are
present in the DNA-protein complex, which might stabilize NFY.
The B Subunit of NFY Binds the Transcriptional Adapter p300 in
Vitro and in Vivo--
Nuclear extract resulted in a larger NFY·DNA
protein complex than did recombinant NFY (Fig. 1, lanes 7 and 8). This effect was detected with both the
oligonucleotide B and the ferritin promoter fragment (data not shown).
Because the nuclear adapter p300 binds the B-binding factor(s) at
region
Next, we expressed in E. coli the epitope-tagged p300
molecule subdivided in three fragments: the N terminus (p300N, residues 1-690), the central region (p300M, residues 671-1194), and the C
terminus (p300C, residues 1135-2414). The recombinant A, B, and C
subunits of NFY were incubated with the three p300 peptides tethered to
flag-M2 affinity gel. Panel A of Fig.
2 shows the immunoblot with the
antibodies against the NFY-subunits mixed with the p300 central region;
the NFY-B subunit specifically binds p300 bound to the column.
Bacterial lysates expressing the vector alone, purified with the same
procedure and blotted with the three antibodies did not show any
specific signal (lanes 2, 4, and 6). The specificity of the binding between recombinant NFY-B and p300 was
confirmed by testing in the same assay a different recombinant protein
(PKA regulatory subunit, RII
These data indicate that the central segment of p300 specifically binds
the NFY-B subunit in vitro. To demonstrate that the selective interaction between p300 and subunit NFY-B occurs also in vivo, we expressed full-length epitope-tagged p300 in
HeLa cells, isolated the protein by affinity chromatography, and
immunoblotted the eluate with the antibodies against the three NFY
subunits. A band of about 30 kDa, corresponding to the size of the B
subunit in vivo (Fig. 3,
lane NE), binds p300, which confirms the data obtained in vitro (compare Figs. 2 and 3). Also, in
vivo subunit NFY-B interacts preferentially with the central
fragment of p300 as shown by the same experiment done with eukaryotic
vectors containing p300 subdivided into the three fragments (data not
shown).
To confirm that the central segment of p300 specifically interacts with
NFY/B, we expressed in HeLa cells the three segments of p300 along with
a ferritin promoter-CAT construct. Fig. 4
shows the result of the CAT assay. The expression of construct p300M strongly reduced the ferritin promoter basal activity. These data suggest that the overexpression of p300 residues 671-1194 titrates NFY and inhibits assemblage of the productive transcriptional complex
on the ferritin promoter. The slight inhibition by the C fragment might
result from titration of RNA polymerase complex or by low affinity
NFY-B binding.
Conclusions--
The region of the ferritin H promoter necessary
for both basal and cAMP-stimulated transcription contains, in position
Many transcription factors depend on the nuclear co-activator proteins
CBP and p300 to recruit RNA polymerase II to specific promoters (for a
review, see Ref. 13). We have recently demonstrated that the active
transcription complex on the
Biochemical analyses of NFY complex demonstrated that subunits B and C
associate through a subdomain that binds the DNA and resembles an
One important conclusion of our work is that the complex mediates the
cAMP-responsiveness of ferritin and other CAAT-containing promoters.
60 region of the
human H ferritin promoter, the B site. DNA binding analysis with
specific antibodies demonstrates that NFY/B/C subunits tightly bind
this site and that NFY/C subunit is masked in vivo by
binding with other protein(s). NFY binds the co-activator p300.
Specifically, the NFY/B subunit interacts with the central segment of
p300 in vivo and in vitro. cAMP substantially
increases the formation of the NFY·p300 complex. Taken together these
data provide a general model of cAMP induction of non-CRE-containing
promoters and suggest that the NFY-B·p300 complex is located at the
5' end of the promoter and the NFY-B·C·TFIIB on the 3' end toward
the transcription start site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
132 nucleotides from the transcription start site, binds the
ubiquitous transcription factor Sp1 and, in basal conditions, accounts
for about 50% of the total transcription of the promoter (1). Element
B is located at position
62 and is constituted by a CAAT box on the
noncoding strand, flanked by a stretch of GC-rich sequences (1).
Element B induces increased transcription in differentiating cells (2)
and in cells exposed to G418 (3). Element B is also the target site of
cAMP signaling on ferritin promoter (4). Specifically, we
previously identified binding activity in nuclear extracts at the
B-box, called Bbf,1 which
also binds the co-activator molecule p300/CBP. We found that cAMP
stimulates assemblage of the Bbf·p300 complex (4). Inhibition of
phosphatases by okadaic acid also induces and/or stabilizes this
functional complex (5).
MATERIALS AND METHODS
antibodies for 1 h at
room temperature. Bound antibodies were detected with anti-rabbit or
anti-mouse horseradish peroxidase-conjugated secondary antibodies
(1:5000) and with ECL (Amersham Pharmacia Biotech).
-D-thiogalactopyranoside for
2 h at 37 °C. Bacteria were lysed by sonication in a lysis buffer; after centrifugation, the supernatant was incubated with anti-flag M2 affinity gel for 1 h at 4 °C. The beads were
collected by centrifugation, washed four times with the lysis buffer,
and incubated with rNF-Y or with rRII
in 100 µl of binding buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM Na3O4V, 50 mM NaF, 0.5% Nonidet P-40) for 1 h at 4 °C. The
beads were collected by centrifugation and washed four times with the
binding buffer. SDS-PAGE sample buffer (20 µl) was added to the
beads; the sample was heated at 95 °C and then loaded on a 12%
SDS-PAGE gel.
100 to +1 of the
human H promoter, terminally labeled. EMSA and competition assays were as described previously (4). Anti-NFY antibodies were assayed as
supershifting reagent; antibodies were added to the nuclear extract and
incubated for 1 h on ice before addition of the labeled probes.
RESULTS
100/
50 region of the human ferritin H promoter
contains a CAAT box, flanked by two GC-rich sequences, on the noncoding strand. This cis-element, the "B-box" (1) or "FF fragment" (12), induces transcription of the H gene (2-4). We asked whether the
CAAT-binding factor NFY, also called "CBF", binds this sequence in
the ferritin promoter. To this end, we incubated a specific B
oligonucleotide with recombinant NFY protein (NFY) and analyzed the
resulting DNA-protein complex by EMSA. As shown in Fig.
1, recombinant NFY formed a specific
complex with the B element of ferritin promoter (lanes
1, 2, and 3). To determine which of the NFY
subunits (A, B, or C) binds the DNA of the B element, we added antibodies against NFY-A, -B, and -C subunits to the incubation mixture. Anti-A, anti-B, and anti-C antibodies abolished the retarded DNA-protein complex (Fig. 1, lanes 4, 5, and
6). We also incubated the B oligonucleotide with nuclear
extract from HeLa cells (lane 8) and probed the mixture with
the anti-NFY antibodies (lanes 9, 10, and
11). The formation of the NFY·DNA complex was prevented by
the antibodies against subunits NFY-A and NFY-B (lanes 9 and 10) but was unaffected by anti C-antibodies (lane
11). The discrepancy between in vivo and in
vitro results suggests that binding to other component(s) masked
the subunit C in the nuclear extracts. This is borne out by the larger
size of the DNA-protein complex generated by the nuclear extract
compared with the recombinant NFY (compare lanes 1 and
8).
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Fig. 1.
NFY binds the B element of the H ferritin
promoter, in position 62. 10 ng of recombinant NFY protein
(lanes 1-7) or 4 µg of nuclear extracts from HeLa cells
(lanes 8-12) were incubated with 0.5 ng of labeled B site
oligonucleotide (B oligo), as described under "Materials
and Methods." Competitions were performed by preincubating the
recombinant protein with a 100-fold molar excess of the unlabeled B
site oligo (spec; lane 2), or with unspecific
oligonucleotide (non spec; lane 3). The
anti-NFY-A (Ab antiA), anti-NFY-B (Ab antiB), and
anti-NFY-C (Ab antiC) antibodies were incubated with the
recombinant NFY protein (lanes 4-6) or with HeLa nuclear
extract (lanes 9-11) for 1 h on ice prior to the
addition of the labeled B oligo. In lanes 7 and
12 is the result of incubation with non-immune antisera
(NS). The lower mobility complex obtained with nuclear
extracts (lanes 8-12) is because of unspecific binding, as
demonstrated by competition experiments (data not shown).
100 to +1 base pairs). Addition of
anti-NFY antibodies to the nuclear extract supershifted the complex
NFYI·DNA (data not shown). Antibodies to subunit C did not alter the
migration of the DNA-protein complexes (data not shown), which
coincides with the data obtained with the oligonucleotide (Fig. 1).
62 of ferritin promoter (4), we tested whether the
heterotrimeric NFY directly interacts with p300. Addition of specific
p300 antibodies to the binding mixture did not cause a reproducible
supershift of the retarded DNA-protein complex (data not shown).
), as shown in lanes 5 and 6 of panel B. Parallel immunoblots with anti-p300
flag (panel B, lane 3) and with the NFY-A, -B, and -C
recombinant subunits (panel C) showed that the amounts of
the reactants in the experiment were comparable. Note that the
recombinant C-subunit is a truncated form with a size of about 25 kDa
in SDS-page. In the same assay, the N- and C-terminal fragments of p300
did not bind any NFY-subunit (data not shown).
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Fig. 2.
The in vitro interaction
between p300 and NFY requires the NFY-B subunit and the 671-1194
region of p300. A, the p300 region from amino acids 671 to
1194, flag-tagged, was expressed in E. coli, and the
resulting bacterial lysate was used in protein-protein interaction
assays as described under "Materials and Methods." Lanes
1, 3, and 5, recombinant NFY
(NFY) mixed with the p300-expressing bacterial lysates.
Lanes 2, 4, and 6, recombinant NFY
(NFY) mixed with the bacterial lysate from mock-transfected
cells. After incubation, the protein-protein complexes were analyzed by
Western blot with anti-NFY-B antibody (Ab antiB; lanes
1 and 2), or with anti-NFY-A antibody (Ab
antiA; lanes 3 and 4), or with anti NFY-C
antibody (Ab antiC; lanes 5 and 6).
B, recombinant NFY (NFY) mixed with the
p300-expressing bacterial lysate (lane 1) or with lysate
from mock-transfected cells (mock; lane 2),
bacterial lysate from flag-tagged-p300 transfected cells
(p300; lane 3), bacterial lysate from
mock-transfected cells (mock; lane 4),
recombinant RII (RII
) mixed with the p300-expressing
bacterial lysate (lane 5), or with lysate from
mock-transfected cells (mock; lane 6). After
incubation, the protein-protein complexes were analyzed by Western blot
with anti-NFY antibody (Ab anti NFY; lanes 1 and
2), or with anti-flag antibody (Ab anti FLAG;
lanes 3 and 4), or with anti-RII
antibody
(Ab anti RII
; lanes 5 and 6).
C, the recombinant proteins NFY-A, NFY-B, and NFY-C were
analyzed on 12% SDS-PAGE followed by Western blot analysis with
anti-NFY-B (lane 1), anti-NFY-A (lane 2), and
anti-NFY-C (lane 3) antibodies.
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Fig. 3.
In vivo interaction between p300
and NFY. HeLa cells were transfected with 5 µg of eukaryotic
p300-expressing plasmid. 1 mg of total cell extract was
immunoprecipitated with anti-p300 antibody (IP p300 +) or
with nonimmune antisera (IP NS +). After electrophoresis on
SDS-PAGE and electroblotting, the membrane containing immobilized
immunocomplex(es) was analyzed by Western blot using anti-NFY antibody
(WB NFY). 50 µg of HeLa nuclear extract were loaded as a
control for the presence of NFY (NE).
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Fig. 4.
p300M, co-transfected in HeLa cells, reduces
transcription driven by the B element of ferritin promoter. CAT
activity driven by the Bbf-TATAA construct (4) in HeLa cells
(Bbf-TATAA) and in HeLa cells co-transfected with the expression
vectors for the central region of p300 (+p300M), for the
p300 C terminus (+p300C), and for the p300 N terminus
(+p300N). The values are the mean of three independent
experiments and are expressed as percentage of the acetylated form of
the 14C-chloramphenicol.
62 relative to the start site, an inverted CAAT box (1, 4). We show
that the B-box is recognized by the heterotrimeric transcription factor
NFY. NFY subunit B binds the central segment of the co-activator p300
in vitro and in vivo. Note that NFY subunits A,
B, and C correspond to CBF subunits B, A, and C, respectively.
62 H promoter contains p300 and that
cAMP stimulates the formation of this complex (4). Protein-protein
binding assays in vivo and in vitro indicate
that NFY is the component of the ferritin transcription complex that specifically interacts with p300 (Figs. 3 and 4). Preincubation of
extracts with protein kinase A (PKA) substantially increased the
endogenous B protein retained by recombinant p300, suggesting that NFY
is the target of PKA (data not shown). Ferritin promoter might provide
a novel structural framework for the induction of transcription of
non-CRE-containing promoters (4, 9, and 10).
-helical structure first identified in the core histone proteins
(H2B/H2A) (14, 15). The B/C histone-fold plays a crucial role in the
creation of a functional NFY-CAAT box binding complex because subunit A
associates only with the B/C heterodimer (7). The B/C heterodimer
associates with the histone acetyltransferase P/CAF (16). The histone
acetyltransferase activity found in NFY immunoprecipitates might derive
directly from p300, which has intrinsic histone acetyltransferase
activity (17) and from associated P/CAF. Note that P/CAF does not
associate with isolated subunit B, whereas p300 does (Ref. 16 and Figs. 2 and 3). Taken together these data suggest a model illustrated in Fig.
5. NFY-B binds the central segment of
p300 and recruits it to the CAAT box with NFY-B and NFY-A. P/CAF
interacts with the NFY-B/C complex and probably is stabilized by p300
and B/C heterodimer binding the DNA (16). This complex possesses
powerful and specific histone acetylase activity (derived from p300 and P/CAF), which by modifying the structure of chromatin facilitates transcription (18). A recent report highlights the role of NFY as key
regulator of acetylation responsiveness for hsp promoter in
Xenopus oocytes by virtue of its interaction with p300 (19). The interaction of p300 with TFIIB positions the complex to the transcription start site (20, 21). At the p300 N terminus other factors
might interacts with upstream sequence elements (CREB or Jun). We have
evidence that c-Jun can trans-activate ferritin promoter by interacting
with the p300·NFY-B complex. We suggest that low affinity DNA sites
can be stabilized by this interaction.2
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Fig. 5.
Schematic diagram of NFY-p300 complex on the
B-site of H ferritin promoter. NFY is indicated by the A, B, and C
letters. J represents an N terminus interacting protein, such as c-Jun
or CREB (13). The pCAF/p300 interaction (20) and the p300/TFIIB
interaction (21) have been described previously.
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FOOTNOTES |
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* This work was supported by grants from MURST (Ministero Università e Ricerca Scientifica e Tecnologica) (Rome) PRIN 1997, from Consiglio Nazionale Ricerche (CNR), targeted project "Biotecnologie," and from Associazione Italiana Ricerca sul Cancro (AIRC).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.
§ Dipartimento di Medicina Sperimentale e Patologia, Università "La Sapienza", Roma, 00161, Italy.
¶ Dept. of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030.
** To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli, Via S. Pansini 5, I-80131 Napoli. Tel.: 39-081-7463647; Fax: 39-081-7463650; E-mail: fsc{at}unicz.it.
2 M. C. Faniello, V. E. Avvedimento, F. Cimino, and F. Costanzo, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Bbf, B-box-binding factor; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; CRE, cAMP-responsive element; EMSA, electrophoretic mobility shift assay; H, heavy; PKA, protein kinase A; PAGE, polyacrylamide gel electrophoresis.
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